Rapid autofluorescence flow cytometric analysis of agonist-induced neutrophil and eosinophil polarization reveals novel insights into 5-oxo-ETE-mediated granulocyte activation

Rapid autofluorescence flow cytometric analysis of agonist-induced neutrophil and eosinophil polarization reveals novel insights into 5-oxo-ETE-mediated granulocyte activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rapid autofluorescence flow cytometric analysis of agonist-induced neutrophil and eosinophil polarization reveals novel insights into 5-oxo-ETE-mediated granulocyte activation Anuruddika J Fernando, Fiona Rossi, Destiny Docherty, Anna Popravko, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7204746/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Nov, 2025 Read the published version in Journal of Inflammation → Version 1 posted 9 You are reading this latest preprint version Abstract Minimizing unintended granulocyte activation while measuring functional responsiveness is essential, as the use of external probes, antibodies, or fluorescent dyes can potentially alter cellular responsiveness. To address this, we employed an antibody-free flow cytometry approach that measures forward scatter (FSC) to detect variations in cell-size, morphology, and shape; some key indicators of neutrophil and eosinophil activation. Human peripheral blood neutrophils, containing contaminating eosinophils, were isolated using discontinuous Percoll gradients and pre-treated with receptor antagonists [e.g., cyclosporin-H (an FPR1 antagonist) and CP105696 (a BLT1 receptor antagonist)] prior to stimulation with agonists such as fMLF (an FPR1 agonist) and LTB 4 (a BLT1 agonist). Imaging flow cytometry, together with FSC analysis, enabled assessment of cell polarization and associated morphological changes. Importantly, autofluorescence-based gating allowed for the identification of contaminating eosinophils within the mixed granulocyte population, allowing parallel assessment of shape-change in both neutrophils and eosinophils in response to the same ligands. Stimulation of neutrophils with fMLF resulted in distinct FSC shifts compared to unstimulated controls across all flow cytometers tested, which were inhibited by cyclosporin-H, but not CP105696. Morphological analysis confirmed these changes corresponded with increased cell area and perimeter and decreased circularity, hallmarks of cell polarisation. Additionally, selective activation of eosinophils (but not neutrophils) by eotaxin, and dual activation of both cell types by the arachidonic acid metabolite 5-oxo-ETE, were confirmed through specific gating strategies. Taken together, these findings support the use of FSC-based flow cytometry as a rapid, scalable and effective method for evaluating granulocyte polarisation and screening candidate therapeutics targeting immune cell activation in disease contexts. granulocytes neutrophils eosinophils shape-change flow-cytometry 5-oxo-ETE Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Granulocytes, primarily neutrophils and eosinophils, are key bone-marrow derived inflammatory cells of the innate immune system and neutrophils, as first responders, are rapidly recruited to sites of infection and tissue injury [ 1 – 3 ]. These immune cells are guided to inflammatory sites by various chemoattractants, including formylated peptides such as N-formylmethionine-leucyl-phenylalanine (fMLF), a potent synthetic peptide agonist which mimics bacterial infection or mitochondrial damage, is known to ligate FPR1 to activate neutrophils [ 4 ]. Similarly, the arachidonic-acid derived mediators like leukotriene B 4 (LTB 4 ) and 5-oxo-ETE, and the cytokine CXCL8 (also known as IL-8) also activate downstream neutrophil functions [ 1 , 5 , 6 ]. These agonists act through binding to specific G-protein coupled receptors (GPCRs); fMLF activates FPR1, LTB 4 binds BLT1, 5-oxo-ETE signals via OXER1, and eotaxin binds CCR3 primarily expressed on eosinophils [ 4 , 7 – 10 ]. Physiologically, granulocyte priming is considered a necessary step for full activation, facilitating receptor expression, degranulation and production of reactive oxygen species (ROS) [ 11 ]. Upon activation, neutrophils and eosinophils initiate key effector functions evolved at neutralising pathogens and resolving tissue damage. However, dysregulated or uncontrolled granulocyte activation can contribute to chronic inflammation and collateral tissue damage [ 4 , 5 ]. Therefore, the possibility of screening pharmaceutical compounds such as receptor antagonists to counteract this activation, could be therapeutically beneficial. Many in vitro assays have been developed to assess downstream granulocyte responses, such as intracellular calcium flux, chemotaxis, degranulation, phagocytosis, ROS generation, and extracellular trap formation. However, these methods often require the use of fluorogenic dyes [(e.g., dihydrorhodamine (DHR) that detects intracellular ROS)], fluorescent antibodies and chemiluminescent probes (e.g., lucigenin detects superoxide anions), which can penetrate cells and may themselves inadvertently activate granulocytes and skew results [ 12 – 14 ]. Furthermore, given how readily granulocytes can be activated, the isolation procedures may themselves stimulate cells, potentially affecting their downstream functions [ 15 , 16 ]. Use of a well-established method combining dextran sedimentation with discontinuous Percoll gradient centrifugation offers high leukocyte purity (≥ 95%), similar to negative selection methods using immunomagnetic beads, while minimising activation of immune cells [ 17 , 18 ]. However, minimal contamination with highly autofluorescent eosinophils in neutrophil preparations has allowed for the distinction between these two cell types using flow cytometry, enabling their use in various applications [ 19 – 21 ]. In this study, we present a simple and reliable antibody-free flow cytometry method for detecting neutrophil and eosinophil activation based on their scatter profiles [forward scatter (FSC) and side scatter (SSC)], which reflect changes in cell size, shape, and granularity. In addition, this technique is compatible with both standard flow cytometers and imaging flow cytometers, offering the added benefit of visualising cell morphology at the single-cell level following activation [ 22 , 23 ]. Although similar approaches have been used in other studies to assess neutrophil activation, we expand on this by incorporating imaging flow cytometry and applying the method to explore previously uncharacterised mechanisms of neutrophil and eosinophil activation, in response to agonists such as eotaxin and 5-oxo-ETE [ 24 , 25 ]. Using this approach, we examined shape changes (quantified by FSC-A; forward scatter-area) in neutrophils and eosinophils in response to specific GPCR agonists (e.g., fMLF and LTB 4 ) and their respective antagonists [e.g., cyclosporin-H (CsH) and CP105696]. Although previous studies have examined the role of LTB 4 and its receptor antagonist in neutrophil shape change and apoptosis [ 26 ], comparative analyses with other receptor agonists and antagonists, such as CsH, have not been reported. Our study addresses this gap by directly comparing these effects and moreover, while morphological changes in primed and activated neutrophils have been described, we employ imaging flow cytometry to visualise these changes in real-time, offering new insights into the dynamic behaviour of neutrophils with detail not previously achieved [ 27 ]. We further analysed the differential effects of 5-oxo-ETE and the chemokine eotaxin on neutrophil and eosinophil cellular polarisation. Multiple analysis strategies were used to assess granulocyte shape change, providing evidence into a rapid and scalable method for screening immune cell responses and identifying potential therapeutic targets. Methods Ethics statement Human blood samples were collected from healthy adult volunteers with informed consent, in accordance with ethical approval granted by the Lothian Research Ethics Committee (EMREC Reference: 21-EMREC-041). All collections were carried out at the Institute for Regeneration and Repair (IRR; University of Edinburgh) by registered phlebotomists, adhering to established institutional and local guidelines. Isolation of human granulocytes from peripheral blood Peripheral human blood was collected into Falcon tubes containing 3.8% sodium citrate (Sigma). Leukocytes were isolated using dextran (Sigma) sedimentation followed by discontinuous Percoll (GE Healthcare) gradient centrifugation. Mononuclear cells were collected from 55/70% interface, while granulocytes were isolated from the 70/81% interface, as described [ 14 , 20 ]. The resulting granulocytes consisting primarily of neutrophils with some (usually less than 5%) eosinophils, were washed with phosphate-buffered saline (PBS) without cations and cell counts were performed using a haemocytometer. Cell purity was assessed by cytocentrifuge preparations, with ≥ 95% of cells identified as neutrophils with minor eosinophil contamination. Cells were resuspended in PBS with cations (Mg 2+ and Ca 2+ ) at the appropriate concentration for downstream assays. Assessment of neutrophil purity To assess cell morphology and determine neutrophil purity, cytocentrifuge preparations were carried out using 1×10 6 granulocytes in 100µL of suspension. Cells were loaded onto cytocentrifuge chambers and centrifuged at 300rpm for 3min using a Thermo Scientific Shandon cytocentrifuge. The resulting cell films were briefly air-dried, fixed in 100% methanol, and subsequently stained with Eosin and Haematoxylin using Diff-Kwik solutions 2 and 3 (Thermo Scientific), respectively. Slides were mounted with DPX mounting medium (Sigma) and examined under a light microscope at 100× magnification. Neutrophil purity was determined by counting at least 100 cells across 10 randomly chosen fields of view, based on standard morphological criteria (e.g., number of nuclear lobes and staining characteristics of the cytoplasm and granules). Granulocyte cell polarisation (shape change assay) To assess the effects of receptor agonists and antagonists on neutrophil activation by measuring shape change, isolated human granulocytes were resuspended at 2×10 6 cells/mL. Cells were pre-incubated with the antagonists, CsH (Sigma) or CP105696 (MedchemExpress), or PBS vehicle control, for 30 minutes at 37°C on a shaking heat block (300rpm). Antagonist concentrations used are provided in the results section or figure legends. Following antagonist incubation, cells were stimulated with receptor-specific agonists; fMLF (Sigma) or LTB 4 (Cayman chemical) for 30 minutes, and eotaxin (Peprotech) or 5-oxo-ETE (Avanti polar lipids) for 2 or 5 minutes at 37°C, based on prior concentration-response and time-course optimisation experiments. Post stimulation, cells were fixed by adding an equal volume of 4% paraformaldehyde (PFA, ChemCruz) in PBS and stored at 4°C until analysis. Neutrophil shape change was assessed by measuring changes in forward scatter (FSC-A) using flow cytometry analysers and sorters such as the BD LSR Fortessa (BD Biosciences) and BD FACS Aria II (BD Biosciences), respectively. Data were analysed with FCSExpress 7 (research edition), and shape-change was quantified based on forward scatter area (FSC-A), including area under the curve derived from histogram width and weight, as previously described [ 28 ]. Imaging flow cytometry In addition to assessing neutrophil activation based on light scatter parameters, isolated neutrophils treated with fMLF, or PBS vehicle control were analysed using imaging flow cytometry on the Thermo Attune Cytpix system (Thermo Scientific). This platform integrates traditional flow cytometry with high-resolution imaging, at the single cell level, enabling detailed morphological assessment. Image analysis was performed on single cells using Attune cytometric software, which quantified neutrophil phenotypic parameters such as circularity, cell area, and perimeter (in microns), in both activated and non-activated neutrophils. Due to inherent autofluorescence of eosinophils, these cells were distinguished from neutrophils using autofluorescence-based gating, as described [ 20 ]. Granulocytes were first gated based on forward and side scatter profiles with doublets excluded using FSC-area and FSC-height parameters. Human eosinophils were differentiated from neutrophils using the 488nm laser (525/50nm filter), in combination with either conventional side scatter profiles or signals from the 355nm laser (450/50nm filter). This gating strategy enabled identification of both cell types within a single plot, allowing quantification of shape-change responses to various agonists and antagonists in neutrophils and eosinophils. Statistical Analyses All data (unless specified) are presented as mean ± standard error of the mean (SEM) based on experimental replicates from individual donors. Flow-cytometry data were analysed using FCSExpress7 (research edition) and visualised using GraphPad Prism (version 9). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc multiple-comparisons test for analyses involving more than two treatment groups. Comparisons related to neutrophil image analysis and shape change measurements from different flow cytometry instruments were assessed using unpaired Student’s t -tests. Statistical significance was interpreted as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****). Results Integrated conventional and imaging flow cytometry provide complementary insights into neutrophil activation, inhibition, and morphological dynamics Shape-changes in response to chemoattractants allow neutrophils to migrate and usually squeeze in between endothelial cells during transmigration to inflammatory sites. To establish and quantify this response, we assessed neutrophil shape change in response to chemoattractants using forward scatter area (FSC-A) as a readout for neutrophil activation. Agonist and antagonist concentrations, as well as incubation times, were optimised through concentration-response and time-course experiments (Supplementary Figs. 1 and 2) and guided by existing literature [ 26 , 29 , 30 ]. Neutrophils isolated from healthy human blood using dextran sedimentation and Percoll gradients were pre-treated with GPCR antagonists such as cyclosporin-H (CsH) and CP10569, prior to agonist stimulation, followed by fixing and analysis using flow cytometry (Fig. 1 A). Stimulation of neutrophils with 100nM fMLF resulted in a significant increase in shape-change compared to unstimulated controls, as detected by an increase in forward scatter area (FSC-A) following gating for granulocytes and singlet populations using a BD FACS Aria II cell sorter (p < 0.05, n = 10; Fig. 1 B& 1 C). The same approach was used to assess inhibition of neutrophil activation using GPCR antagonists. Cyclosporin-H and CP105696 alone did not induce neutrophil shape-change (data not shown), but each selectively inhibited the corresponding receptor-mediated response. CsH significantly reduced fMLF-induced neutrophil shape change but had no effect on LTB 4 -induced activation, whereas CP105696 significantly inhibited LTB 4 -mediated shape change but did not affect fMLF-induced response. These results highlight the use of this method in studying both receptor-specific activation and inhibition of neutrophils using defined ligands. Furthermore, although fMLF can result in secondary LTB 4 production, the lack of effect of CP105696 on fMLF-induced shape change confirms that this response is not mediated via the LTB 4 /BLT1 pathway [ 26 , 31 , 32 ]. Developments in flow-cytometry have enabled real-time visualization of individual cells and assessment of morphological features alongside fluorescence-based measurements [ 25 ]. To validate neutrophil shape changes observed by forward scatter (FSC-A), we used the Attune Cytpix imaging flow cytometer to directly image neutrophils following stimulation with fMLF or PBS, and in the presence of cyclosporin-H. Using the Attune Cytometric software (v6.21), hundreds of cells per sample were analysed. Unstimulated neutrophils exhibited a rounded morphology, while fMLF-stimulated cells displayed elongated and irregular shapes consistent with activation-induced morphological changes (Fig. 2 A). Neutrophil morphological parameters such as circularity, perimeter, and area were quantified in cells from three donors under unstimulated conditions, following fMLF stimulation, and after pre-incubation with CsH. fMLF stimulation significantly increased cell perimeter and area, while reducing circularity, consistent with forward scatter findings (Fig. 2 B-D). Pre-treatment with CsH effectively inhibited these fMLF-induced morphological changes, restoring neutrophil shape to control-like levels. In addition, CsH alone did not cause any detectable changes in neutrophil morphology (data not shown). These findings support the use of FSC-A as a reliable indicator of neutrophil shape change and activation, providing a simple, scalable approach to assess polarisation and morphological changes in response to chemoattractants or pharmacological modulators. Neutrophil activation induces forward scatter increases in different flow cytometer sorters and analysers Neutrophil shape-changes, reflected by increased forward light scatter (FSC-A), were detectable using flow cytometer sorters (e.g., BD FACS Aria II) and analysers (e.g., BD LSR Fortessa, Attune NXT). Forward versus side scatter plots from each instrument demonstrated consistent increases in FSC-A following 100nM fMLF stimulation, indicating increased neutrophil shape and size alteration (Figs. 3 A&B). However, differences in the distribution of FSC values on histogram plots were observed between instruments. This variability is influenced by the position and design of the obscuration bar, which in some analysers shifts the FSC histogram peak to the right, while others produce a wider distribution or shifts in the opposite direction (Fig. 3 C). Differences in data analysis between flow cytometry analysers and sorters, as well as the choice of statistical metrics, are important considerations when interpreting neutrophil shape change results. Mean fluorescent intensity (MFI) and forward scatter area standard deviation (FSC-A SD) are commonly used metrics to represent these changes. MFI reflects the fluorescent intensity of neutrophils within the singlet gate comparing stimulated versus unstimulated cells; however, its values vary depending on the instrument. For example, MFI increases with fMLF stimulation when measured on sorters where FSC-A histograms shift to the right, but decreases on some analysers like the LSR Fortessa, where FSC-A shifts to the left. Thus, MFI is relative and highly dependent on instrument settings and calibration. In contrast, FSC-A SD consistently provided more robust measurements of neutrophil shape change, accurately reflecting increases in cell size and morphological heterogeneity across the cell population. An increase in FSC-A SD was observed in fMLF-stimulated neutrophils compared to controls across all instruments tested (n = 5), supporting its use as a preferred metric for this assay (Figs. 3 D and 4 A). These findings thus demonstrate that both flow cytometry analysers and cell sorters can reliably detect stimuli-induced shape changes in cells, despite differences in scatter profiles due to instrumental design features. Neutrophil shape change was also assessed using classical histogram-based methods, in which printed histograms of control and stimulated samples were physically cut, weighed, and their lateral width measured along the x-axis to compare distribution shifts [ 28 ]. Similarly, the histogram weights provide an estimate of the population shift in forward scatter, offering a simple yet effective method to compare activation-induced changes. These approaches produced results consistent with those obtained from flow cytometry sorters and analysers, showing clear distinctions between control and fMLF-stimulated neutrophils (Fig. 4 B). Autofluorescence-based shape change analysis demonstrates eotaxin-specific eosinophil activation profiles To enable downstream assays, it was essential to isolate pure populations of neutrophils and eosinophils from mixed donor granulocyte preparations. Given the lack of specific surface markers to separate these two cell types without affecting their function, a previously described autofluorescence-based sorting strategy was used (20). This method considers the intrinsic differences in autofluorescent properties between neutrophils and eosinophils under specific flow cytometry laser settings. Clear separation between neutrophils and eosinophils was achieved based on their autofluorescence profiles in the 488nm (525/50nm) and 355nm (450/50nm) detectors. In addition, cytocentrifuge preparations of sorted cells from the respective gates confirmed high purity of both cell populations, as verified by H&E (Haematoxylin and Eosin) staining (Fig. 5A). To evaluate the effect of human eotaxin on donor-derived neutrophils and eosinophils, forward scatter-based shape change assays were performed. Compared to fMLF, which robustly activated neutrophils, eotaxin selectively induced shape change in eosinophils but not neutrophils at concentrations of 10 and 100ng/mL (n = 6) (Figs. 5B and 5C). These findings highlight the use of forward scatter analysis for studying selective immune cell activation in response to specific agonists. Additionally, time-course experiments (data not shown) and histogram profiles of forward scatter data showed that eotaxin-mediated eosinophil activation occurs rapidly, within 2–5 minutes, whereas fMLF-induced neutrophil activation was observed at approximately 30 minutes (Figs. 5B and 5C). Figure 5: Eotaxin selectively triggers morphological changes in eosinophils but not neutrophils. (A) Human granulocytes isolated using the dextran/Percoll method were analysed by flow cytometry, revealing a predominantly neutrophil population with minimal eosinophil contamination. Eosinophils (green arrow) were distinguishable from neutrophils (black arrow) based on their distinct autofluorescence. Representative micrographs of sorted neutrophils and eosinophils are shown. (B) Using the established gating strategy, the effect of human eotaxin on neutrophil and eosinophil shape change was assessed. Overlay histograms demonstrate that fMLF (100nM) induces shape change in both cell types, while eotaxin (10ng/mL) selectively activates eosinophils, with no effect on neutrophils. (C) Graphical summary of shape change responses in neutrophils and eosinophils treated with eotaxin (10 and 100ng/mL) for 2 and 5 minutes, based on time-course experiments using cells from six independent donors (n = 6). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; one-way ANOVA with Tukey’s multiple comparisons test was used to assess differences between treatment groups. 5-oxo-ETE induces rapid and reversible shape-change in both neutrophils and eosinophils via forward-scatter profiling To further evaluate the use of this method in assessing granulocyte activation, we investigated the effects of additional lipid mediators on neutrophil and eosinophil shape change. Using forward scatter (FSC-A) profiles as a readout, we compared the activation potential of 5-oxo-ETE, a known eicosanoid associated with inflammation, to that of fMLF and eotaxin. This method allowed us to determine the relative specificity and activation kinetics in both human neutrophils and eosinophil populations. 5-oxo-ETE was evaluated for its ability to induce shape change in human granulocytes. At a concentration of 100nM, it triggered forward scatter increases in both neutrophils and eosinophils, indicating cellular activation (Fig. 6 A). While the response in eosinophils was comparable to that seen with fMLF, neutrophils showed a weaker response relative to fMLF stimulation. Similar to eotaxin, 5-oxo-ETE induced a rapid and transient shape change response in both cell types, peaking within 2 minutes and declining by 5min (n = 5) (Fig. 6 B). Discussion This study represents a simple, rapid and scalable method to reliably detect granulocyte activation in response to inflammatory stimuli, through changes in cell morphology, using forward scatter (FSC-A) and autofluorescence properties by flow cytometry. Our findings support the use of FSC-A shifts as a readout for both neutrophil and eosinophil activation, providing a label-free method to detect shape changes in these immune cells. During an infection or sterile injury, neutrophil activation occurs when pathogen and damage-derived formylated peptides bind the G-protein coupled receptor FPR1, triggering calcium flux and downstream functions such as chemotaxis, phagocytosis, degranulation, ROS production and formation of extracellular traps [ 4 , 33 ]. While these responses aid host defence, dysregulated activation contributes to various inflammatory diseases. Therefore, targeting receptor signalling through modulation of GPCR signalling to limit immune cell activation could offer therapeutic benefit. The synthetic peptide, fMLF, used in this study is a well-established FPR1 agonist and is often used to model neutrophil activation in vitro [ 4 , 33 ]. Similarly, lipid mediators like LTB 4 can activate neutrophils via the GPCR BLT1, and formylated peptides can also act as priming agents to promote release of LTB 4 and IL-8, to further amplify the inflammatory response [ 6 ]. Although previous studies have examined the role of LTB4 and its receptor antagonist in neutrophil shape change and apoptosis, comparative analyses with other receptor agonists and antagonists, such as CsH, have not been reported. Our study addresses this gap by directly comparing these effects and moreover, while morphological changes in primed and activated neutrophils have been described, we employ imaging flow cytometry to visualise these changes in real-time, offering new insights into the dynamic behaviour of neutrophils with detail not previously achieved. One of the main advantages of this approach is its adaptability across different flow cytometry instruments. Although the shape and size of FSC-A histograms vary between analysers and sorters used, we have successfully shown that the overall outcome in shape change remains consistent across different instruments. The variability in FSC-A histograms is due to the position and design of the obscuration bar, which in some analysers shift the FSC peak to the right, while in others produce a wider distribution or shift in the opposite direction. The obscuration bar, positioned in front of the FSC detection lens, reduces stray light by blocking unwanted scatter, allowing only light scattered at specific angles to reach the FSC detector [ 34 ]. This enhances FSC resolution by minimizing background noise. Therefore, this work highlights the robustness of the assay, while also emphasising the importance of using standardised statistical parameters such as FSC-A SD instead of MFI (based on instrument settings and calibration), to account for differences in instrument calibration and alignment. In addition, we confirm previous findings that granulocyte populations, in particular neutrophils and eosinophils can easily be distinguished using their inherent autofluorescence properties by flow cytometry. This antibody-free approach allows clean separation between the cell types using intrinsic cellular autofluorescence properties induced by the 488 nm and 355 nm lasers, without the need for surface staining, which was further validated using H&E stains of sorted cells. In addition, this method can be applied using even the most basic flow cytometers by relying on side scatter profiles when the ultraviolet laser is not available. This approach is further useful for assessing functional responses, where antibody binding could potentially influence cell behaviour. Fixation was carried out only when long-term storage was required; otherwise, unfixed cells were used immediately and could be maintained on ice for 10–30 minutes, as previously described, to preserve cellular integrity [ 20 ]. To this end, our results show that both fMLF and 5-oxo-ETE induce shape change or morphological activation in neutrophils and eosinophils, while eotaxin selectively activates eosinophils which is consistent with published receptor biology, where fMLF acts via FPR1/FPR2, 5-oxo-ETE via OXER1, and eotaxin via CCR3, expressed on eosinophils [ 4 , 35 , 36 ]. Interestingly, the rapid kinetics observed with both 5-oxo-ETE and eotaxin further indicates that this assay is suitable for assessing early activation of immune cells. For the first time, the data also show that granulocyte responses to 5-oxo-ETE and eotaxin are transient, peaking between 2 and 5 minutes and declining thereafter. This pattern is consistent with the in vivo granulocyte behaviour, where shape change precedes transmigration to inflammatory sites [ 37 ]. Interestingly, eotaxin acts a negative regulator for neutrophil activation and recruitment, in response to inflammatory challenges either through altering their responsiveness to cytokines or reducing adhesion and transmigration, as seen in a mouse model of endotoxemia [ 38 ]. In addition, eosinophil activation with eotaxin has previously been shown to enhance neutrophil responses through LTB 4 and 5-oxo-ETE production via 5-lipoxygenase (5-LOX) pathway, thereby suggesting a possible feedback mechanism [ 39 , 40 ]. This further highlights a role for eosinophils in secondary neutrophil recruitment. The speed, scalability, cost-effectiveness, and simplicity combined with a framework suitable for high-throughput screening make this method highly suitable for early-stage drug discovery in models of inflammatory disease. However, certain limitations of the method should be acknowledged. Forward scatter data are essentially dependent on instrument optics, and autofluorescence gating of eosinophils and neutrophils may be influenced by differences in granule content in disease states. While shape change is a strong indicator of activation, it does not incorporate all aspects of granulocyte functional responses such as ROS production, degranulation, or cytokine release. Hence, it is a valuable technique that should be combined with complementary assays for in-depth assessment. In brief, this study incorporates a rapid, robust, and versatile method for detecting activation-induced shape changes in granulocytes. Furthermore, this technique provides valuable insights into immune cell behaviour and a practical tool for mechanistic studies and drug screening. Conclusions This study presents a robust, scalable and label-free methodology to assess changes in granulocyte activation through shape-change analysis in response to different agonists and antagonists using scatter and autofluorescent properties. The approach allows effective distinction between neutrophil and eosinophil responses to the same agonist, offering a valuable tool for screening pharmacological agents targeting immune cell activation. Our Findings reveal that eotaxin selectively activates eosinophils, but not neutrophils, while 5-oxox-ETE induces shape change in both cell types, highlighting their distinctive responsiveness. Interestingly both agonists trigger rapid and reversible activation, indicating their relevance in acute inflammatory processes. Overall, the method provides a platform for studying granulocyte biology and identifying candidate therapeutics to mitigate immune-driven inflammation. Abbreviations fMLF= N-formylmethionine-leucyl-phenylalanine LTB 4 = Leukotriene B 4 IL-8= Interleukin-8 ROS= Reactive oxygen species FSC=Forward scatter SSC=Side scatter GPCR= G-protein coupled receptor 5-oxo-ETE= 5-oxo-eicosatetraenoic acid Declarations Acknowledgements We thank Claire Cryer and Ailsa Laird from the Institute for Regeneration and Repair Flow Cytometry & Cell Sorting Facility for their helpful advice and support. We thank Adiso Therapeutics Inc. (USA) and the University of Edinburgh for funding AJF and the Medical Research Council UK for the Programme grant (MR/K013386/1) for AGR. Authors’ contributions AJF and AGR: Conceptualization, Investigation, Data curation, Formal analysis, Methodology, Writing-original draft. FR, DD, AP, LM and BH: Investigation, Data curation, formal analysis. AGR, KD and RG: Supervision, Funding acquisition and Editing. All authors contributed to manuscript revisions and approved the final version. Funding This work was supported by a PhD studentship jointly funded by the University of Edinburgh and Adiso Therapeutics Inc., and by the Medical Research Council UK through a Programme Grant (MR/K013386/1). Data and materials availability Data supporting the findings of this study are available in the supplementary section and from the corresponding author upon reasonable request. Reagents and materials used in the study can also be provided upon request, subject to material transfer agreements where applicable. Consent for publication Not applicable Competing interests The authors declare no competing interests References Shim HB, Deniset JF, Kubes P. Neutrophils in homeostasis and tissue repair. Int Immunol. 2022;34(8):399-407. Potey PM, Rossi AG, Lucas CD, Dorward DA. Neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential. J Pathol. 2019;247(5):672-85. 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Son K, Mukherjee M, McIntyre BAS, Eguez JC, Radford K, LaVigne N, et al. Improved recovery of functionally active eosinophils and neutrophils using novel immunomagnetic technology. J Immunol Methods. 2017;449:44-55. Weil GJ, Chused TM. Eosinophil autofluorescence and its use in isolation and analysis of human eosinophils using low microfluorometry. Blood. 1981;57(6):1099-104. Dorward DA, Lucas CD, Alessandri AL, Marwick JA, Rossi F, Dransfield I, et al. Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production. J Leukoc Biol. 2013;94(1):193-202. Sabroe I, Hartnell A, Jopling LA, Bel S, Ponath PD, Pease JE, et al. Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways. J Immunol. 1999;162(5):2946-55. Depince-Berger AE, Aanei C, Iobagiu C, Jeraiby M, Lambert C. New tools in cytometry. Morphologie. 2016;100(331):199-209. Manohar SM, Shah P, Nair A. Flow cytometry: principles, applications and recent advances. Bioanalysis. 2021;13(3):181-98. O’Flaherty JT, Cordes J, Redman J, Thomas MJ. 5-Oxo-Eicosatetraenoate, a Potent Human Neutrophil Stimulus. Biochemical and Biophysical Research Communications. 1993;192(1):129-34. Willetts L, Ochkur SI, Jacobsen EA, Lee JJ, Lacy P. Eosinophil Shape Change and Secretion. In: Walsh GM, editor. Eosinophils: Methods and Protocols. New York, NY: Springer New York; 2014. p. 111-28. 596 29 597 Murray J, Ward C, O'Flaherty JT, Dransfield I, Haslett C, Chilvers ER, et al. Role of leukotrienes in the regulation of human granulocyte behaviour: dissociation between agonist-induced activation and retardation of apoptosis. Br J Pharmacol. 2003;139(2):388-98. Bashant KR, Vassallo A, Herold C, Berner R, Menschner L, Subburayalu J, et al. Real time deformability cytometry reveals sequential contraction and expansion during neutrophil priming. J Leukoc Biol. 2019;105(6):1143-53. Dimbat M, Porter PE, Stross FH. Apparatus Requirements for Quantitative Applications. Analytical Chemistry. 1956;28(3):290-7. Dorward DA, Lucas CD, Doherty MK, Chapman GB, Scholefield EJ, Conway Morris A, et al. Novel role for endogenous mitochondrial formylated peptide-driven formyl peptide receptor 1 signalling in acute respiratory distress syndrome. Thorax. 2017;72(10):928-36. Burke-Gaffney A, Hellewell PG. Eotaxin stimulates eosinophil adhesion to human lung microvascular endothelial cells. Biochem Biophys Res Commun. 1996;227(1):35-40. Plagge M, Laskay T. Early Production of the Neutrophil-Derived Lipid Mediators LTB(4) and LXA(4) Is Modulated by Intracellular Infection with Leishmania major. Biomed Res Int. 2017;2017:2014583. Hidi R, Coëffier E, Vargaftig BB. Formation of LTB4 by fMLP-stimulated alveolar macrophages accounts for eosinophil migration in vitro. J Leukoc Biol. 1992;51(5):425-31. Prossnitz ER, Ye RD. The N-formyl peptide receptor: a model for the study of chemoattractant receptor structure and function. Pharmacol Ther. 1997;74(1):73-102. Arkesteijn GJA, Lozano-Andrés E, Libregts S, Wauben MHM. Improved Flow Cytometric Light Scatter Detection of Submicron-Sized Particles by Reduction of Optical Background Signals. Cytometry A. 2020;97(6):610-9. Conroy DM, Williams TJ. Eotaxin and the attraction of eosinophils to the asthmatic lung. Respir Res. 2001;2(3):150-6. Powell WS, Rokach J. 5-Oxo-ETE and Inflammation. In: Steinhilber D, editor. Lipoxygenases in Inflammation. Cham: Springer International Publishing; 2016. p. 185-210. Beesley JE, Pearson JD, Hutchings A, Carleton JS, Gordon JL. Granulocyte migration through endothelium in culture. J Cell Sci. 1979;38:237-48. Cheng SS, Lukacs NW, Kunkel SL. Eotaxin/CCL11 is a negative regulator of neutrophil recruitment in a murine model of endotoxemia. Exp Mol Pathol. 2002;73(1):1-8. Kitaura M, Nakajima T, Imai T, Harada S, Combadiere C, Tiffany HL, et al. Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J Biol Chem. 1996;271(13):7725-30. Luz RA, Xavier-Elsas P, de Luca B, Masid-de-Brito D, Cauduro PS, Arcanjo LC, et al. 5-lipoxygenase-dependent recruitment of neutrophils and macrophages by eotaxin-stimulated murine eosinophils. Mediators Inflamm. 2014;2014:102160. Additional Declarations No competing interests reported. Supplementary Files SupplementaryData.docx Cite Share Download PDF Status: Published Journal Publication published 06 Nov, 2025 Read the published version in Journal of Inflammation → Version 1 posted Editorial decision: Revision requested 01 Sep, 2025 Reviews received at journal 01 Sep, 2025 Reviews received at journal 05 Aug, 2025 Reviewers agreed at journal 04 Aug, 2025 Reviewers agreed at journal 01 Aug, 2025 Reviewers invited by journal 01 Aug, 2025 Editor assigned by journal 01 Aug, 2025 Submission checks completed at journal 01 Aug, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7204746","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508615497,"identity":"097abe23-b0f9-440b-bf61-9ffaad0e21a6","order_by":0,"name":"Anuruddika J Fernando","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Anuruddika","middleName":"J","lastName":"Fernando","suffix":""},{"id":508615498,"identity":"99aa0b68-c1d0-48a1-a822-883581b70a54","order_by":1,"name":"Fiona Rossi","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Fiona","middleName":"","lastName":"Rossi","suffix":""},{"id":508615499,"identity":"5b0335d0-588c-4e96-80fd-c883af07595b","order_by":2,"name":"Destiny Docherty","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Destiny","middleName":"","lastName":"Docherty","suffix":""},{"id":508615500,"identity":"95717bd7-f635-4feb-a9c7-06cfbeb5fa76","order_by":3,"name":"Anna Popravko","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Popravko","suffix":""},{"id":508615505,"identity":"912ff36b-87a8-4059-98ee-1882d212ff1b","order_by":4,"name":"Lucy Masters","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Lucy","middleName":"","lastName":"Masters","suffix":""},{"id":508615508,"identity":"1967fcce-5533-46f6-9de9-fe41ebe72bab","order_by":5,"name":"Boydd Houston","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Boydd","middleName":"","lastName":"Houston","suffix":""},{"id":508615510,"identity":"8722f8be-f200-4274-b010-42ea3bbb7ccf","order_by":6,"name":"Renu Gupta","email":"","orcid":"","institution":"Adiso Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Renu","middleName":"","lastName":"Gupta","suffix":""},{"id":508615511,"identity":"84734222-f0b9-42dc-bb17-79d5d84040bb","order_by":7,"name":"Kevin Dhaliwal","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Dhaliwal","suffix":""},{"id":508615513,"identity":"0979ed20-90f7-4ade-b421-ce5cceb56e88","order_by":8,"name":"Adriano G. Rossi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIie2RsQrCMBRFXwnE5VHXhIL+QkSQTu2vpBQ6OTg6SCkIcfObIgVdKl07WvoDTqWTSLo4JqNDzhTCO+HePACP5w+hQF5kModFBWy+QouyBCqIGaKoHRVeIcwKMOmoCG2UY5mGfLh1cEpANNpBmZo6U1GRx3DPQTwrq3IgvdKSRvsdA6pBtJZgqUZBNqpMKW9HBh8HRRiFKxIohpQFSjsEq6mo0XTBYhtn1xy5tf7j3A/mx9aXuu/eY7IKG2lJRgB+r0r7Vjwej8fjwhd9EDyxEsHAGQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Edinburgh","correspondingAuthor":true,"prefix":"","firstName":"Adriano","middleName":"G.","lastName":"Rossi","suffix":""}],"badges":[],"createdAt":"2025-07-24 10:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7204746/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7204746/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12950-025-00472-8","type":"published","date":"2025-11-06T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90893428,"identity":"1591ac54-b881-438c-9924-ad52c3c1b497","added_by":"auto","created_at":"2025-09-09 11:24:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":428288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003efMLF and LTB\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003etrigger human neutrophil shape change, detected by FSC-A and cell morphological alterations. \u003c/strong\u003e(A) Schematic of the human neutrophil shape change assay. Neutrophils were isolated by dextran sedimentation followed by Percoll gradient centrifugation, then incubated with PBS or receptor antagonists prior to agonist stimulation (e.g., fMLF). Cells were analysed following fixation by flow cytometry. (B) Gating strategy and representative histogram plots showing no activation following PBS treatment, while fMLF stimulation induced shape changes detectable as shifts in forward-scatter (FSC-A) on a BD FACSAria II. (C) Quantification of human neutrophil shape change in response to fMLF compared to PBS across ten independent donor experiments (n=10). (D) Flow cytometry histograms and overlays demonstrating receptor-specific inhibition of neutrophil shape change (e.g., CP105696 inhibits LTB\u003csub\u003e4 \u003c/sub\u003e-mediated activation), indicating blockade of agonist-induced responses. (E) fMLF-induced shape change (100nM) was inhibited by CsH (10µM), while CP105696 (10µM) blocked LTB\u003csub\u003e4\u003c/sub\u003e (100nM)-induced shape change but not fMLF-induced responses, confirming specific inhibition of FPR1 and BLT1 receptors.\u003csub\u003e \u003c/sub\u003e****p≤0.0001, unpaired t-test with Welch’s correction (PBS vs. fMLF); one-way ANOVA with Tukey’s post hoc test for multiple comparisons between treatment groups.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/ecf1b4bb9187686db731e3fc.png"},{"id":90897237,"identity":"decf520a-86ce-4f37-a005-52fe1de3187d","added_by":"auto","created_at":"2025-09-09 11:40:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":593505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophil activation induces morphological changes detectable by flow cytometry, which were inhibited by CsH treatment.\u003c/strong\u003e Imaging flow cytometry was used to assess changes in cell shape and size during neutrophil activation, alongside forward scatter analysis. (A) Representative brightfield images showing neutrophil elongation and irregular morphology following stimulation with fMLF (100nM), which was inhibited by CsH (10µM), reverting to a morphology similar to unstimulated controls. (B-D) Quantitative morphological analysis using the Attune Cytpix imaging flow cytometer and it’s software, showed that fMLF stimulation increased neutrophil area and perimeter, while reducing circularity. These changes were inhibited by CsH treatment. n≥100 cells per condition were analysed across 3 independent experiments. *p≤0.05, **p≤0.01; one way ANOVA with Tukey’s post hoc test. Data are presented as Mean ± SEM.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/9a0fc9b8c058ec73ce156728.png"},{"id":90897238,"identity":"b971c769-f1bf-42fc-8b82-81f2665e87ce","added_by":"auto","created_at":"2025-09-09 11:40:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":590864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFSC profiles of human neutrophil shape change varies depending on the flow cytometry platform used. \u003c/strong\u003e(A) Flow cytometry plots showing human neutrophils treated with PBS and analysed on different flow cytometry platforms (BD FACSCanto II, BD Accuri C6, BD LSR Fortessa, and Attune NXT). (B) Plots of neutrophils stimulated with 100nM fMLF for 30 minutes show a rightward shift or broadening of the FSC-A histogram, depending on the instrument used. (C) Histogram overlays comparing PBS-treated and fMLF-stimulated neutrophils highlight instrument-specific differences in FSC-A profiles, with sorters typically showing a rightward shift. (D)Quantitative analysis of neutrophil shape change based on the standard deviation of FSC-A (FSC-A SD) following PBS and fMLF treatment (n≥3). **p≤0.01, ****p≤0.0001, unpaired t-test with Welch’s correction used to compare forward scatter between PBS control and fMLF-treated cells.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/28d986a8624aede96c8a4522.png"},{"id":90893430,"identity":"d27950ac-5f46-46a3-b3a0-61e488c2d0f0","added_by":"auto","created_at":"2025-09-09 11:24:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":248851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophil activation is measurable by physical histogram weighing and width analysis across multiple flow cytometers. \u003c/strong\u003e(A) Graphical analysis of neutrophil activation by fMLF (100nM) versus control using the Attune Nxt flow cytometer. Data are presented using histogram statistics, including mean fluorescent intensity (MFI) and standard deviation (SD) (n≥4). (B) Alternative representation of activation data using histogram widths or area under the curve obtained by physically weighing carefully cut histograms. Data from multiple flow cytometers are shown for both control and fMLF-treated neutrophils for comparison (n=3). *p≤0.05, ***p≤0.001, ****p≤0.0001; unpaired t-test with Welch’s correction comparing PBS control and fMLF-treated groups.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/aad0e61ee7823540c73b458d.png"},{"id":90899020,"identity":"eed227b6-4e1b-49b3-b540-19292b4bd3fb","added_by":"auto","created_at":"2025-09-09 11:56:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":641550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEotaxin selectively triggers morphological changes in eosinophils but not neutrophils. \u003c/strong\u003e(A) Human granulocytes isolated using the dextran/Percoll method were analysed by flow cytometry, revealing a predominantly neutrophil population with minimal eosinophil contamination. Eosinophils (green arrow) were distinguishable from neutrophils (black arrow) based on their distinct autofluorescence. Representative micrographs of sorted neutrophils and eosinophils are shown. (B) Using the established gating strategy, the effect of human eotaxin on neutrophil and eosinophil shape change was assessed. Overlay histograms demonstrate that fMLF (100nM) induces shape change in both cell types, while eotaxin (10ng/mL) selectively activates eosinophils, with no effect on neutrophils. (C) Graphical summary of shape change responses in neutrophils and eosinophils treated with eotaxin (10 and 100ng/mL) for 2 and 5 minutes, based on time-course experiments using cells from six independent donors (n=6). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001; one-way ANOVA with Tukey’s multiple comparisons test was used to assess differences between treatment groups.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/91eb86ce1e5c722103fc8db4.png"},{"id":90897245,"identity":"6ea9e7d3-6d2f-49e4-aad7-a65e196727d1","added_by":"auto","created_at":"2025-09-09 11:40:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":181968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e5-oxo-eicosatetraenoic acid induces shape change in both human neutrophils and eosinophils \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) Flow cytometry analysis shows that 5-oxo-ETE (100nM), a lipid mediator, induces rapid shape change in human eosinophils, with peak activation at 2 minutes post-stimulation. The observed response is comparable to that seen with fMLF (100nM). In neutrophils, 5-oxo-ETE also triggers shape change, evident by increased forward scatter, though the effect is less than that induced by fMLF. (B) Quantitative representation of shape change in neutrophils and eosinophils, following stimulation with 100nM 5-oxo-ETE for 2 and 5 minutes, based on time-course experiments using cells from five individual donors (n=5). *p≤0.05, **p≤0.01; one-way ANOVA with Tukey’s multiple comparisons test comparing treatment groups.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/5aa76b1b2127b2c6ef97524b.png"},{"id":95564825,"identity":"b08221df-5f37-4bf2-babc-b5b1968faee0","added_by":"auto","created_at":"2025-11-10 16:10:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3545290,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/3718ec8f-5219-47a7-a6c8-2756afe5ac2c.pdf"},{"id":90897236,"identity":"17039065-718a-4837-a5e0-95007b975357","added_by":"auto","created_at":"2025-09-09 11:40:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":385001,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7204746/v1/66c13f835c23bfd12e892410.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rapid autofluorescence flow cytometric analysis of agonist-induced neutrophil and eosinophil polarization reveals novel insights into 5-oxo-ETE-mediated granulocyte activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGranulocytes, primarily neutrophils and eosinophils, are key bone-marrow derived inflammatory cells of the innate immune system and neutrophils, as first responders, are rapidly recruited to sites of infection and tissue injury [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These immune cells are guided to inflammatory sites by various chemoattractants, including formylated peptides such as N-formylmethionine-leucyl-phenylalanine (fMLF), a potent synthetic peptide agonist which mimics bacterial infection or mitochondrial damage, is known to ligate FPR1 to activate neutrophils [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Similarly, the arachidonic-acid derived mediators like leukotriene B\u003csub\u003e4\u003c/sub\u003e (LTB\u003csub\u003e4\u003c/sub\u003e) and 5-oxo-ETE, and the cytokine CXCL8 (also known as IL-8) also activate downstream neutrophil functions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These agonists act through binding to specific G-protein coupled receptors (GPCRs); fMLF activates FPR1, LTB\u003csub\u003e4\u003c/sub\u003e binds BLT1, 5-oxo-ETE signals via OXER1, and eotaxin binds CCR3 primarily expressed on eosinophils [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e–\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Physiologically, granulocyte priming is considered a necessary step for full activation, facilitating receptor expression, degranulation and production of reactive oxygen species (ROS) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Upon activation, neutrophils and eosinophils initiate key effector functions evolved at neutralising pathogens and resolving tissue damage. However, dysregulated or uncontrolled granulocyte activation can contribute to chronic inflammation and collateral tissue damage [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, the possibility of screening pharmaceutical compounds such as receptor antagonists to counteract this activation, could be therapeutically beneficial.\u003c/p\u003e\u003cp\u003eMany \u003cem\u003ein vitro\u003c/em\u003e assays have been developed to assess downstream granulocyte responses, such as intracellular calcium flux, chemotaxis, degranulation, phagocytosis, ROS generation, and extracellular trap formation. However, these methods often require the use of fluorogenic dyes [(e.g., dihydrorhodamine (DHR) that detects intracellular ROS)], fluorescent antibodies and chemiluminescent probes (e.g., lucigenin detects superoxide anions), which can penetrate cells and may themselves inadvertently activate granulocytes and skew results [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, given how readily granulocytes can be activated, the isolation procedures may themselves stimulate cells, potentially affecting their downstream functions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Use of a well-established method combining dextran sedimentation with discontinuous Percoll gradient centrifugation offers high leukocyte purity (≥ 95%), similar to negative selection methods using immunomagnetic beads, while minimising activation of immune cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, minimal contamination with highly autofluorescent eosinophils in neutrophil preparations has allowed for the distinction between these two cell types using flow cytometry, enabling their use in various applications [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e–\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we present a simple and reliable antibody-free flow cytometry method for detecting neutrophil and eosinophil activation based on their scatter profiles [forward scatter (FSC) and side scatter (SSC)], which reflect changes in cell size, shape, and granularity. In addition, this technique is compatible with both standard flow cytometers and imaging flow cytometers, offering the added benefit of visualising cell morphology at the single-cell level following activation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Although similar approaches have been used in other studies to assess neutrophil activation, we expand on this by incorporating imaging flow cytometry and applying the method to explore previously uncharacterised mechanisms of neutrophil and eosinophil activation, in response to agonists such as eotaxin and 5-oxo-ETE [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUsing this approach, we examined shape changes (quantified by FSC-A; forward scatter-area) in neutrophils and eosinophils in response to specific GPCR agonists (e.g., fMLF and LTB\u003csub\u003e4\u003c/sub\u003e) and their respective antagonists [e.g., cyclosporin-H (CsH) and CP105696]. Although previous studies have examined the role of LTB\u003csub\u003e4\u003c/sub\u003e and its receptor antagonist in neutrophil shape change and apoptosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], comparative analyses with other receptor agonists and antagonists, such as CsH, have not been reported. Our study addresses this gap by directly comparing these effects and moreover, while morphological changes in primed and activated neutrophils have been described, we employ imaging flow cytometry to visualise these changes in real-time, offering new insights into the dynamic behaviour of neutrophils with detail not previously achieved [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We further analysed the differential effects of 5-oxo-ETE and the chemokine eotaxin on neutrophil and eosinophil cellular polarisation. Multiple analysis strategies were used to assess granulocyte shape change, providing evidence into a rapid and scalable method for screening immune cell responses and identifying potential therapeutic targets.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eEthics statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHuman blood samples were collected from healthy adult volunteers with informed consent, in accordance with ethical approval granted by the Lothian Research Ethics Committee (EMREC Reference: 21-EMREC-041). All collections were carried out at the Institute for Regeneration and Repair (IRR; University of Edinburgh) by registered phlebotomists, adhering to established institutional and local guidelines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of human granulocytes from peripheral blood\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePeripheral human blood was collected into Falcon tubes containing 3.8% sodium citrate (Sigma). Leukocytes were isolated using dextran (Sigma) sedimentation followed by discontinuous Percoll (GE Healthcare) gradient centrifugation. Mononuclear cells were collected from 55/70% interface, while granulocytes were isolated from the 70/81% interface, as described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The resulting granulocytes consisting primarily of neutrophils with some (usually less than 5%) eosinophils, were washed with phosphate-buffered saline (PBS) without cations and cell counts were performed using a haemocytometer. Cell purity was assessed by cytocentrifuge preparations, with ≥ 95% of cells identified as neutrophils with minor eosinophil contamination. Cells were resuspended in PBS with cations (Mg\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e) at the appropriate concentration for downstream assays.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of neutrophil purity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess cell morphology and determine neutrophil purity, cytocentrifuge preparations were carried out using 1×10\u003csup\u003e6\u003c/sup\u003e granulocytes in 100µL of suspension. Cells were loaded onto cytocentrifuge chambers and centrifuged at 300rpm for 3min using a Thermo Scientific Shandon cytocentrifuge. The resulting cell films were briefly air-dried, fixed in 100% methanol, and subsequently stained with Eosin and Haematoxylin using Diff-Kwik solutions 2 and 3 (Thermo Scientific), respectively. Slides were mounted with DPX mounting medium (Sigma) and examined under a light microscope at 100× magnification. Neutrophil purity was determined by counting at least 100 cells across 10 randomly chosen fields of view, based on standard morphological criteria (e.g., number of nuclear lobes and staining characteristics of the cytoplasm and granules).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGranulocyte cell polarisation (shape change assay)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the effects of receptor agonists and antagonists on neutrophil activation by measuring shape change, isolated human granulocytes were resuspended at 2×10\u003csup\u003e6\u003c/sup\u003e cells/mL. Cells were pre-incubated with the antagonists, CsH (Sigma) or CP105696 (MedchemExpress), or PBS vehicle control, for 30 minutes at 37°C on a shaking heat block (300rpm). Antagonist concentrations used are provided in the results section or figure legends. Following antagonist incubation, cells were stimulated with receptor-specific agonists; fMLF (Sigma) or LTB\u003csub\u003e4\u003c/sub\u003e (Cayman chemical) for 30 minutes, and eotaxin (Peprotech) or 5-oxo-ETE (Avanti polar lipids) for 2 or 5 minutes at 37°C, based on prior concentration-response and time-course optimisation experiments. Post stimulation, cells were fixed by adding an equal volume of 4% paraformaldehyde (PFA, ChemCruz) in PBS and stored at 4°C until analysis. Neutrophil shape change was assessed by measuring changes in forward scatter (FSC-A) using flow cytometry analysers and sorters such as the BD LSR Fortessa (BD Biosciences) and BD FACS Aria II (BD Biosciences), respectively. Data were analysed with FCSExpress 7 (research edition), and shape-change was quantified based on forward scatter area (FSC-A), including area under the curve derived from histogram width and weight, as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eImaging flow cytometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn addition to assessing neutrophil activation based on light scatter parameters, isolated neutrophils treated with fMLF, or PBS vehicle control were analysed using imaging flow cytometry on the Thermo Attune Cytpix system (Thermo Scientific). This platform integrates traditional flow cytometry with high-resolution imaging, at the single cell level, enabling detailed morphological assessment. Image analysis was performed on single cells using Attune cytometric software, which quantified neutrophil phenotypic parameters such as circularity, cell area, and perimeter (in microns), in both activated and non-activated neutrophils.\u003c/p\u003e\u003cp\u003eDue to inherent autofluorescence of eosinophils, these cells were distinguished from neutrophils using autofluorescence-based gating, as described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Granulocytes were first gated based on forward and side scatter profiles with doublets excluded using FSC-area and FSC-height parameters. Human eosinophils were differentiated from neutrophils using the 488nm laser (525/50nm filter), in combination with either conventional side scatter profiles or signals from the 355nm laser (450/50nm filter). This gating strategy enabled identification of both cell types within a single plot, allowing quantification of shape-change responses to various agonists and antagonists in neutrophils and eosinophils.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll data (unless specified) are presented as mean ± standard error of the mean (SEM) based on experimental replicates from individual donors. Flow-cytometry data were analysed using FCSExpress7 (research edition) and visualised using GraphPad Prism (version 9). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc multiple-comparisons test for analyses involving more than two treatment groups. Comparisons related to neutrophil image analysis and shape change measurements from different flow cytometry instruments were assessed using unpaired Student’s \u003cem\u003et\u003c/em\u003e-tests. Statistical significance was interpreted as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIntegrated conventional and imaging flow cytometry provide complementary insights into neutrophil activation, inhibition, and morphological dynamics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eShape-changes in response to chemoattractants allow neutrophils to migrate and usually squeeze in between endothelial cells during transmigration to inflammatory sites. To establish and quantify this response, we assessed neutrophil shape change in response to chemoattractants using forward scatter area (FSC-A) as a readout for neutrophil activation. Agonist and antagonist concentrations, as well as incubation times, were optimised through concentration-response and time-course experiments (Supplementary Figs.\u0026nbsp;1 and 2) and guided by existing literature [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Neutrophils isolated from healthy human blood using dextran sedimentation and Percoll gradients were pre-treated with GPCR antagonists such as cyclosporin-H (CsH) and CP10569, prior to agonist stimulation, followed by fixing and analysis using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eStimulation of neutrophils with 100nM fMLF resulted in a significant increase in shape-change compared to unstimulated controls, as detected by an increase in forward scatter area (FSC-A) following gating for granulocytes and singlet populations using a BD FACS Aria II cell sorter (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026amp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The same approach was used to assess inhibition of neutrophil activation using GPCR antagonists. Cyclosporin-H and CP105696 alone did not induce neutrophil shape-change (data not shown), but each selectively inhibited the corresponding receptor-mediated response. CsH significantly reduced fMLF-induced neutrophil shape change but had no effect on LTB\u003csub\u003e4\u003c/sub\u003e-induced activation, whereas CP105696 significantly inhibited LTB\u003csub\u003e4\u003c/sub\u003e-mediated shape change but did not affect fMLF-induced response. These results highlight the use of this method in studying both receptor-specific activation and inhibition of neutrophils using defined ligands. Furthermore, although fMLF can result in secondary LTB\u003csub\u003e4\u003c/sub\u003e production, the lack of effect of CP105696 on fMLF-induced shape change confirms that this response is not mediated via the LTB\u003csub\u003e4\u003c/sub\u003e/BLT1 pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDevelopments in flow-cytometry have enabled real-time visualization of individual cells and assessment of morphological features alongside fluorescence-based measurements [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To validate neutrophil shape changes observed by forward scatter (FSC-A), we used the Attune Cytpix imaging flow cytometer to directly image neutrophils following stimulation with fMLF or PBS, and in the presence of cyclosporin-H. Using the Attune Cytometric software (v6.21), hundreds of cells per sample were analysed. Unstimulated neutrophils exhibited a rounded morphology, while fMLF-stimulated cells displayed elongated and irregular shapes consistent with activation-induced morphological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Neutrophil morphological parameters such as circularity, perimeter, and area were quantified in cells from three donors under unstimulated conditions, following fMLF stimulation, and after pre-incubation with CsH. fMLF stimulation significantly increased cell perimeter and area, while reducing circularity, consistent with forward scatter findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). Pre-treatment with CsH effectively inhibited these fMLF-induced morphological changes, restoring neutrophil shape to control-like levels. In addition, CsH alone did not cause any detectable changes in neutrophil morphology (data not shown). These findings support the use of FSC-A as a reliable indicator of neutrophil shape change and activation, providing a simple, scalable approach to assess polarisation and morphological changes in response to chemoattractants or pharmacological modulators.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNeutrophil activation induces forward scatter increases in different flow cytometer sorters and analysers\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNeutrophil shape-changes, reflected by increased forward light scatter (FSC-A), were detectable using flow cytometer sorters (e.g., BD FACS Aria II) and analysers (e.g., BD LSR Fortessa, Attune NXT). Forward versus side scatter plots from each instrument demonstrated consistent increases in FSC-A following 100nM fMLF stimulation, indicating increased neutrophil shape and size alteration (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026amp;B). However, differences in the distribution of FSC values on histogram plots were observed between instruments. This variability is influenced by the position and design of the obscuration bar, which in some analysers shifts the FSC histogram peak to the right, while others produce a wider distribution or shifts in the opposite direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eDifferences in data analysis between flow cytometry analysers and sorters, as well as the choice of statistical metrics, are important considerations when interpreting neutrophil shape change results. Mean fluorescent intensity (MFI) and forward scatter area standard deviation (FSC-A SD) are commonly used metrics to represent these changes. MFI reflects the fluorescent intensity of neutrophils within the singlet gate comparing stimulated versus unstimulated cells; however, its values vary depending on the instrument. For example, MFI increases with fMLF stimulation when measured on sorters where FSC-A histograms shift to the right, but decreases on some analysers like the LSR Fortessa, where FSC-A shifts to the left. Thus, MFI is relative and highly dependent on instrument settings and calibration. In contrast, FSC-A SD consistently provided more robust measurements of neutrophil shape change, accurately reflecting increases in cell size and morphological heterogeneity across the cell population. An increase in FSC-A SD was observed in fMLF-stimulated neutrophils compared to controls across all instruments tested (n\u0026thinsp;=\u0026thinsp;5), supporting its use as a preferred metric for this assay (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These findings thus demonstrate that both flow cytometry analysers and cell sorters can reliably detect stimuli-induced shape changes in cells, despite differences in scatter profiles due to instrumental design features.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNeutrophil shape change was also assessed using classical histogram-based methods, in which printed histograms of control and stimulated samples were physically cut, weighed, and their lateral width measured along the x-axis to compare distribution shifts [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Similarly, the histogram weights provide an estimate of the population shift in forward scatter, offering a simple yet effective method to compare activation-induced changes. These approaches produced results consistent with those obtained from flow cytometry sorters and analysers, showing clear distinctions between control and fMLF-stimulated neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAutofluorescence-based shape change analysis demonstrates eotaxin-specific eosinophil activation profiles\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo enable downstream assays, it was essential to isolate pure populations of neutrophils and eosinophils from mixed donor granulocyte preparations. Given the lack of specific surface markers to separate these two cell types without affecting their function, a previously described autofluorescence-based sorting strategy was used (20). This method considers the intrinsic differences in autofluorescent properties between neutrophils and eosinophils under specific flow cytometry laser settings. Clear separation between neutrophils and eosinophils was achieved based on their autofluorescence profiles in the 488nm (525/50nm) and 355nm (450/50nm) detectors. In addition, cytocentrifuge preparations of sorted cells from the respective gates confirmed high purity of both cell populations, as verified by H\u0026amp;E (Haematoxylin and Eosin) staining (Fig.\u0026nbsp;5A).\u003c/p\u003e\u003cp\u003eTo evaluate the effect of human eotaxin on donor-derived neutrophils and eosinophils, forward scatter-based shape change assays were performed. Compared to fMLF, which robustly activated neutrophils, eotaxin selectively induced shape change in eosinophils but not neutrophils at concentrations of 10 and 100ng/mL (n\u0026thinsp;=\u0026thinsp;6) (Figs.\u0026nbsp;5B and 5C). These findings highlight the use of forward scatter analysis for studying selective immune cell activation in response to specific agonists. Additionally, time-course experiments (data not shown) and histogram profiles of forward scatter data showed that eotaxin-mediated eosinophil activation occurs rapidly, within 2\u0026ndash;5 minutes, whereas fMLF-induced neutrophil activation was observed at approximately 30 minutes (Figs.\u0026nbsp;5B and 5C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure 5: Eotaxin selectively triggers morphological changes in eosinophils but not neutrophils.\u003c/b\u003e (A) Human granulocytes isolated using the dextran/Percoll method were analysed by flow cytometry, revealing a predominantly neutrophil population with minimal eosinophil contamination. Eosinophils (green arrow) were distinguishable from neutrophils (black arrow) based on their distinct autofluorescence. Representative micrographs of sorted neutrophils and eosinophils are shown. (B) Using the established gating strategy, the effect of human eotaxin on neutrophil and eosinophil shape change was assessed. Overlay histograms demonstrate that fMLF (100nM) induces shape change in both cell types, while eotaxin (10ng/mL) selectively activates eosinophils, with no effect on neutrophils. (C) Graphical summary of shape change responses in neutrophils and eosinophils treated with eotaxin (10 and 100ng/mL) for 2 and 5 minutes, based on time-course experiments using cells from six independent donors (n\u0026thinsp;=\u0026thinsp;6). *p\u0026thinsp;\u0026le;\u0026thinsp;0.05, **p\u0026thinsp;\u0026le;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026le;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; one-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test was used to assess differences between treatment groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003e5-oxo-ETE induces rapid and reversible shape-change in both neutrophils and eosinophils via forward-scatter profiling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further evaluate the use of this method in assessing granulocyte activation, we investigated the effects of additional lipid mediators on neutrophil and eosinophil shape change. Using forward scatter (FSC-A) profiles as a readout, we compared the activation potential of 5-oxo-ETE, a known eicosanoid associated with inflammation, to that of fMLF and eotaxin. This method allowed us to determine the relative specificity and activation kinetics in both human neutrophils and eosinophil populations.\u003c/p\u003e\u003cp\u003e5-oxo-ETE was evaluated for its ability to induce shape change in human granulocytes. At a concentration of 100nM, it triggered forward scatter increases in both neutrophils and eosinophils, indicating cellular activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). While the response in eosinophils was comparable to that seen with fMLF, neutrophils showed a weaker response relative to fMLF stimulation. Similar to eotaxin, 5-oxo-ETE induced a rapid and transient shape change response in both cell types, peaking within 2 minutes and declining by 5min (n\u0026thinsp;=\u0026thinsp;5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study represents a simple, rapid and scalable method to reliably detect granulocyte activation in response to inflammatory stimuli, through changes in cell morphology, using forward scatter (FSC-A) and autofluorescence properties by flow cytometry. Our findings support the use of FSC-A shifts as a readout for both neutrophil and eosinophil activation, providing a label-free method to detect shape changes in these immune cells. During an infection or sterile injury, neutrophil activation occurs when pathogen and damage-derived formylated peptides bind the G-protein coupled receptor FPR1, triggering calcium flux and downstream functions such as chemotaxis, phagocytosis, degranulation, ROS production and formation of extracellular traps [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. While these responses aid host defence, dysregulated activation contributes to various inflammatory diseases. Therefore, targeting receptor signalling through modulation of GPCR signalling to limit immune cell activation could offer therapeutic benefit. The synthetic peptide, fMLF, used in this study is a well-established FPR1 agonist and is often used to model neutrophil activation \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, lipid mediators like LTB\u003csub\u003e4\u003c/sub\u003e can activate neutrophils via the GPCR BLT1, and formylated peptides can also act as priming agents to promote release of LTB\u003csub\u003e4\u003c/sub\u003e and IL-8, to further amplify the inflammatory response [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although previous studies have examined the role of LTB4 and its receptor antagonist in neutrophil shape change and apoptosis, comparative analyses with other receptor agonists and antagonists, such as CsH, have not been reported. Our study addresses this gap by directly comparing these effects and moreover, while morphological changes in primed and activated neutrophils have been described, we employ imaging flow cytometry to visualise these changes in real-time, offering new insights into the dynamic behaviour of neutrophils with detail not previously achieved.\u003c/p\u003e\u003cp\u003eOne of the main advantages of this approach is its adaptability across different flow cytometry instruments. Although the shape and size of FSC-A histograms vary between analysers and sorters used, we have successfully shown that the overall outcome in shape change remains consistent across different instruments. The variability in FSC-A histograms is due to the position and design of the obscuration bar, which in some analysers shift the FSC peak to the right, while in others produce a wider distribution or shift in the opposite direction. The obscuration bar, positioned in front of the FSC detection lens, reduces stray light by blocking unwanted scatter, allowing only light scattered at specific angles to reach the FSC detector [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This enhances FSC resolution by minimizing background noise. Therefore, this work highlights the robustness of the assay, while also emphasising the importance of using standardised statistical parameters such as FSC-A SD instead of MFI (based on instrument settings and calibration), to account for differences in instrument calibration and alignment.\u003c/p\u003e\u003cp\u003eIn addition, we confirm previous findings that granulocyte populations, in particular neutrophils and eosinophils can easily be distinguished using their inherent autofluorescence properties by flow cytometry. This antibody-free approach allows clean separation between the cell types using intrinsic cellular autofluorescence properties induced by the 488 nm and 355 nm lasers, without the need for surface staining, which was further validated using H\u0026amp;E stains of sorted cells. In addition, this method can be applied using even the most basic flow cytometers by relying on side scatter profiles when the ultraviolet laser is not available. This approach is further useful for assessing functional responses, where antibody binding could potentially influence cell behaviour. Fixation was carried out only when long-term storage was required; otherwise, unfixed cells were used immediately and could be maintained on ice for 10\u0026ndash;30 minutes, as previously described, to preserve cellular integrity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo this end, our results show that both fMLF and 5-oxo-ETE induce shape change or morphological activation in neutrophils and eosinophils, while eotaxin selectively activates eosinophils which is consistent with published receptor biology, where fMLF acts via FPR1/FPR2, 5-oxo-ETE via OXER1, and eotaxin via CCR3, expressed on eosinophils [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Interestingly, the rapid kinetics observed with both 5-oxo-ETE and eotaxin further indicates that this assay is suitable for assessing early activation of immune cells.\u003c/p\u003e\u003cp\u003eFor the first time, the data also show that granulocyte responses to 5-oxo-ETE and eotaxin are transient, peaking between 2 and 5 minutes and declining thereafter. This pattern is consistent with the \u003cem\u003ein vivo\u003c/em\u003e granulocyte behaviour, where shape change precedes transmigration to inflammatory sites [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Interestingly, eotaxin acts a negative regulator for neutrophil activation and recruitment, in response to inflammatory challenges either through altering their responsiveness to cytokines or reducing adhesion and transmigration, as seen in a mouse model of endotoxemia [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, eosinophil activation with eotaxin has previously been shown to enhance neutrophil responses through LTB\u003csub\u003e4\u003c/sub\u003e and 5-oxo-ETE production via 5-lipoxygenase (5-LOX) pathway, thereby suggesting a possible feedback mechanism [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This further highlights a role for eosinophils in secondary neutrophil recruitment.\u003c/p\u003e\u003cp\u003eThe speed, scalability, cost-effectiveness, and simplicity combined with a framework suitable for high-throughput screening make this method highly suitable for early-stage drug discovery in models of inflammatory disease. However, certain limitations of the method should be acknowledged. Forward scatter data are essentially dependent on instrument optics, and autofluorescence gating of eosinophils and neutrophils may be influenced by differences in granule content in disease states. While shape change is a strong indicator of activation, it does not incorporate all aspects of granulocyte functional responses such as ROS production, degranulation, or cytokine release. Hence, it is a valuable technique that should be combined with complementary assays for in-depth assessment.\u003c/p\u003e\u003cp\u003eIn brief, this study incorporates a rapid, robust, and versatile method for detecting activation-induced shape changes in granulocytes. Furthermore, this technique provides valuable insights into immune cell behaviour and a practical tool for mechanistic studies and drug screening.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study presents a robust, scalable and label-free methodology to assess changes in granulocyte activation through shape-change analysis in response to different agonists and antagonists using scatter and autofluorescent properties. The approach allows effective distinction between neutrophil and eosinophil responses to the same agonist, offering a valuable tool for screening pharmacological agents targeting immune cell activation. Our Findings reveal that eotaxin selectively activates eosinophils, but not neutrophils, while 5-oxox-ETE induces shape change in both cell types, highlighting their distinctive responsiveness. Interestingly both agonists trigger rapid and reversible activation, indicating their relevance in acute inflammatory processes. Overall, the method provides a platform for studying granulocyte biology and identifying candidate therapeutics to mitigate immune-driven inflammation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003efMLF= N-formylmethionine-leucyl-phenylalanine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLTB\u003csub\u003e4\u003c/sub\u003e= Leukotriene B\u003csub\u003e4\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIL-8= Interleukin-8\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROS= Reactive oxygen species\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFSC=Forward scatter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSSC=Side scatter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGPCR= G-protein coupled receptor\u003c/p\u003e\n\u003cp\u003e5-oxo-ETE= 5-oxo-eicosatetraenoic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Claire Cryer and Ailsa Laird from the Institute for Regeneration and Repair Flow Cytometry \u0026amp; Cell Sorting Facility for their helpful advice and support. We thank Adiso Therapeutics Inc. (USA) and the University of Edinburgh for funding AJF and \u0026nbsp;the Medical Research Council UK for the Programme grant (MR/K013386/1) for AGR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAJF and AGR: Conceptualization, Investigation, Data curation, Formal analysis, Methodology, Writing-original draft. FR, DD, AP, LM and BH: Investigation, Data curation, formal analysis. AGR, KD and RG: Supervision, Funding acquisition and Editing. All authors contributed to manuscript revisions and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a PhD studentship jointly funded by the University of Edinburgh and Adiso Therapeutics Inc., and by the Medical Research Council UK through a Programme Grant (MR/K013386/1).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available in the supplementary section and from the corresponding author upon reasonable request. Reagents and materials used in the study can also be provided upon request, subject to material transfer agreements where applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShim HB, Deniset JF, Kubes P. Neutrophils in homeostasis and tissue repair. Int Immunol. 2022;34(8):399-407. \u003c/li\u003e\n\u003cli\u003ePotey PM, Rossi AG, Lucas CD, Dorward DA. Neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential. J Pathol. 2019;247(5):672-85. \u003c/li\u003e\n\u003cli\u003eWechsler ME, Munitz A, Ackerman SJ, Drake MG, Jackson DJ, Wardlaw AJ, et al. 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J Cell Sci. 1979;38:237-48. \u003c/li\u003e\n\u003cli\u003eCheng SS, Lukacs NW, Kunkel SL. Eotaxin/CCL11 is a negative regulator of neutrophil recruitment in a murine model of endotoxemia. Exp Mol Pathol. 2002;73(1):1-8. \u003c/li\u003e\n\u003cli\u003eKitaura M, Nakajima T, Imai T, Harada S, Combadiere C, Tiffany HL, et al. Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J Biol Chem. 1996;271(13):7725-30. \u003c/li\u003e\n\u003cli\u003eLuz RA, Xavier-Elsas P, de Luca B, Masid-de-Brito D, Cauduro PS, Arcanjo LC, et al. 5-lipoxygenase-dependent recruitment of neutrophils and macrophages by eotaxin-stimulated murine eosinophils. Mediators Inflamm. 2014;2014:102160.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jinf","sideBox":"Learn more about [Journal of Inflammation](http://journal-inflammation.biomedcentral.com/)","snPcode":"12950","submissionUrl":"https://submission.nature.com/new-submission/12950/3","title":"Journal of Inflammation","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"granulocytes, neutrophils, eosinophils, shape-change, flow-cytometry, 5-oxo-ETE","lastPublishedDoi":"10.21203/rs.3.rs-7204746/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7204746/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMinimizing unintended granulocyte activation while measuring functional responsiveness is essential, as the use of external probes, antibodies, or fluorescent dyes can potentially alter cellular responsiveness. To address this, we employed an antibody-free flow cytometry approach that measures forward scatter (FSC) to detect variations in cell-size, morphology, and shape; some key indicators of neutrophil and eosinophil activation. Human peripheral blood neutrophils, containing contaminating eosinophils, were isolated using discontinuous Percoll gradients and pre-treated with receptor antagonists [e.g., cyclosporin-H (an FPR1 antagonist) and CP105696 (a BLT1 receptor antagonist)] prior to stimulation with agonists such as fMLF (an FPR1 agonist) and LTB\u003csub\u003e4\u003c/sub\u003e (a BLT1 agonist). Imaging flow cytometry, together with FSC analysis, enabled assessment of cell polarization and associated morphological changes. Importantly, autofluorescence-based gating allowed for the identification of contaminating eosinophils within the mixed granulocyte population, allowing parallel assessment of shape-change in both neutrophils and eosinophils in response to the same ligands. Stimulation of neutrophils with fMLF resulted in distinct FSC shifts compared to unstimulated controls across all flow cytometers tested, which were inhibited by cyclosporin-H, but not CP105696. Morphological analysis confirmed these changes corresponded with increased cell area and perimeter and decreased circularity, hallmarks of cell polarisation. Additionally, selective activation of eosinophils (but not neutrophils) by eotaxin, and dual activation of both cell types by the arachidonic acid metabolite 5-oxo-ETE, were confirmed through specific gating strategies. Taken together, these findings support the use of FSC-based flow cytometry as a rapid, scalable and effective method for evaluating granulocyte polarisation and screening candidate therapeutics targeting immune cell activation in disease contexts.\u003c/p\u003e","manuscriptTitle":"Rapid autofluorescence flow cytometric analysis of agonist-induced neutrophil and eosinophil polarization reveals novel insights into 5-oxo-ETE-mediated granulocyte activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 11:24:05","doi":"10.21203/rs.3.rs-7204746/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-01T14:41:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-01T14:39:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-05T15:18:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111024701153516981376168974167620683389","date":"2025-08-04T18:16:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248019242257285268527973985488505415790","date":"2025-08-01T11:12:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-01T10:38:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-01T09:39:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T09:36:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Inflammation","date":"2025-07-24T10:38:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jinf","sideBox":"Learn more about [Journal of Inflammation](http://journal-inflammation.biomedcentral.com/)","snPcode":"12950","submissionUrl":"https://submission.nature.com/new-submission/12950/3","title":"Journal of Inflammation","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d7ab998-9b1f-44cd-bab9-7bb78cc25c94","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:09:43+00:00","versionOfRecord":{"articleIdentity":"rs-7204746","link":"https://doi.org/10.1186/s12950-025-00472-8","journal":{"identity":"journal-of-inflammation","isVorOnly":false,"title":"Journal of Inflammation"},"publishedOn":"2025-11-06 15:57:35","publishedOnDateReadable":"November 6th, 2025"},"versionCreatedAt":"2025-09-09 11:24:05","video":"","vorDoi":"10.1186/s12950-025-00472-8","vorDoiUrl":"https://doi.org/10.1186/s12950-025-00472-8","workflowStages":[]},"version":"v1","identity":"rs-7204746","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7204746","identity":"rs-7204746","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}