Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies

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Müller, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3262595/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Monocytes as well as downstream macrophages and dendritic cells are essential players of the immune system fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies in reporter models, mouse monocytes are divided into a classical and a non-classical subset based on their inversely correlating surface expression of Ly6C and CX3CR1. Here, we analysed the expression of CX3CR1 by flow cytometry using several validated fluorochrome-coupled CX3CR1 antibodies and compared them with the reporter gene signal of a Cx3cr1 GFP reporter mouse strain as well as of tamoxifen-inducible Cx3cr1 reporter mice. Although we were able to validate the specificity of several fluorochrome-coupled CX3CR1 flow cytometry antibodies, mouse Ly6C high classical and Ly6C low non-classical monocytes showed no differences in CX3CR1 expression levels in peripheral blood and spleen, when stained with these antibodies. To the contrary, in reporter mice, we were able to reproduce the inverse correlation of CX3CR1 reporter gene signal and Ly6C surface expression. As determined by qPCR, the Cx3cr1 mRNA expression correlated with the GFP-reporter gene expression as quantified by flow cytometry. In conclusion, our data suggest that there is differential transcription, but not surface expression of CX3CR1 between classical and non-classical monocytes, which limits the suitability of CX3CR1 for phenotyping monocyte subsets by antibody staining. Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages Biological sciences/Immunology/Chemokines Classical monocytes non-classical monocytes C-X3-C Motif Chemokine Receptor 1 (CX3CR1) Lymphocyte antigen 6C2 (Ly6C) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Monocytes are widely conserved cells of the myeloid linage. In the peripheral blood of mice, they account up to 4% of all leucocytes [ 1 , 2 ] and develop from hematopoietic stem cells (HSCs) in the bone marrow via the common myeloid progenitor [ 3 ] . They are recruited from the bloodstream to surrounding tissue where they can differentiate into different macrophages or dendritic cells (DC) dependent on the tissue environment [ 4 ] . Monocytes are known to play a central role in both innate and adaptive immunity. They support and maintain tissue homeostasis by promoting the immune tolerance, contribute to the anti-microbial defence and are essential players of tissue repair and wound healing [ 2 , 5 – 8 ] . However, monocytes are often considered as double-edged sword as they also contribute to the pathogenesis and progression of chronic inflammatory conditions like inflammatory bowel disease, rheumatoid arthritis or multiple sclerosis [ 9 – 13 ] . Over time, our understanding of monocytes has evolved from viewing them as a homogeneous population simply being macrophage precursors to a heterogeneous population with various functions [ 14 , 15 ] . The first evidence of different monocyte subsets was provided by Geissmann et al. identifying a CX3CR1 high and a CX3CR1 low subset with different phenotypic and functional properties, which were confirmed in later reports [ 15 , 16 ] . CX3CR1 is a 7-transmembrane receptor coupled to heterotrimeric G proteins that is important for the adhesion of leukocytes, cell survival and the recruitment of immune cell subpopulations [ 17 ] . Several studies demonstrated that CX3CR1 signalling is an essential survival factor of monocytes [ 18 , 19 ] . Importantly, these initial landmark studies were based on the use of reporter mouse models. Subsequent studies furthermore identified Ly6C as specific marker for discriminating monocytes in two phenotypically and functionally different subtypes [ 20 ] . Conventionally, driven by these studies, mouse monocytes are divided into a classical and a non-classical subset based on their inverse correlation of CX3CR1 and Ly6C surface expression. Classical monocytes, also called inflammatory monocytes, are defined as Ly6C high and CX3CR1 low and are recruited to sites of inflammation at higher rates, where they recognize and phagocytose pathogens and are able to attract other immune cells by secreting cytokines and antimicrobial factors. On the other hand, non-classical monocytes, defined as Ly6C low CX3CR1 high , are characterized by their ability to patrol along the vascular endothelium, to remove cell debris and to promote tissue repair [ 21 , 22 ] . In this study, we aimed to reproduce and validate conventional monocyte subtyping in WT and reporter mice. Therefore, we used Cx3cr1 GFP reporter mice, in which the Cx3cr1 gene is replaced by a green fluorescent protein (GFP) reporter gene. Heterozygous Cx3cr1 +/GFP mice with GFP substitution in only one Cx3cr1 allele were used as reporters to identify Cx3cr1 -expressing monocytes whereas homozygous Cx3cr1 GFP/GFP mice show a functional CX3CR1 knock-out [ 23 ] . Comparing reporter signal and several validated fluorochrome-coupled antibodies head-to-head, we show for the first time that there is a discrepancy between the CX3CR1 reporter gene signal and CX3CR1 surface expression as detected by flow cytometry. However, on mRNA levels, we observed indeed a higher expression of Cx3cr1 in non-classical as compared to classical monocytes in peripheral blood and spleen. These data suggest that CX3CR1 antibody surface staining should be used with caution to profile classical and non-classical monocytes. Results Monocytes from the peripheral blood and spleen of C57Bl/6 mice show no inverse correlation of Ly6C and CX3CR1 expression by flow cytometry In a first step, we aimed to set up a flow cytometry panel to characterise classical and non-classical monocytes based on Ly6C and CX3CR1 as suggested in the literature [ 1 , 24 – 26 ] . Thus, we isolated cells from the peripheral blood of C57Bl/6 mice and analysed them by flow cytometry. Monocytes were defined as Cd11b + Ly6G − Cd115 + (Fig. 1 a). In addition, these cells were also Cd172a + and SiglecF − excluding contamination with type 1 conventional dendritic cells (which are Cd172a − ) and neutrophils (which are SiglecF + ) ( Supplemental Fig. 1a ). In the next step, we used Ly6C (HK1.4, BioLegend) and CX3CR1 (QA16A03, BioLegend) to subcluster these monocytes. While there was a clear separation into Ly6C high and Ly6C low monocytes, we did not observe any relevant differences in the CX3CR1 expression of these monocytes (Fig. 1 b). Similar observations were made using splenocytes from C57Bl/6 mice (Fig. 1 c). Collectively, these data put the inverse correlation of the surface expression of Ly6C and CX3CR1 on mouse monocytes into question. Discrepancy between fluorescent reporter and antibody-based assessment of CX3CR1 expression As the initial studies identifying classical and non-classical monocytes using CX3CR1 were based on reporter mouse models [ 15 , 23 ] , we also investigated the surface expression of CX3CR1 and Ly6C in the peripheral blood of Cx3cr1 GFP mice by flow cytometry. Therefore, we used heterozygous Cx3cr1 GFP/+ mice, which express Cx3cr1 on one allele and GFP under the control of the Cx3cr1 promoter on the other allele. First, we explored whether we can reproduce the inverse correlation of Ly6C and CX3CR1 expression on monocytes based on the CX3CR1 reporter signal. Indeed, we were able to distinguish a Ly6C high CX3CR1 GFP − low and a Ly6C low CX3CR1 GFP − high population in accordance to previous reports (Fig. 2 a) [ 27 – 29 ] . However, using a CX3CR1 antibody (Z8-50, PE, BD Bioscience), we were not able to distinguish these populations on peripheral blood monocytes in the same Cx3cr1 GFP/+ mice (Fig. 2 a, b). Consistently, we did not observe any correlation between antibody-based CX3CR1 and Ly6C expression nor between CX3CR1 antibody and reporter signal (Fig. 2 b). Thus, in a next step, we aimed to verify that the antibody used is indeed functional and able to detect CX3CR1. To this end, we took advantage of different haplotypes of the Cx3cr1 GFP reporter mice. As expected, C57Bl/6 WT and Cx3cr1 +/+ mice showed no expression of CX3CR1-GFP, but similar expression of CX3CR1 as detected by antibody staining on peripheral blood monocytes. In contrast, monocytes from heterozygous Cx3cr1 GFP/+ mice showed expression of CX3CR1-GFP as well as CX3CR1 antibody staining, which was less intense than on C57Bl/6 WT and Cx3cr1 +/+ monocytes. Monocytes from homozygous Cx3cr1 GFP/GFP mice also expressed CX3CR1-GFP (and more intense than Cx3cr1 GFP/+ counterparts), but there was no CX3CR1 antibody staining as expected due to the functional knockout of CX3CR1 (Fig. 2 c, d). Importantly, we did not observe any differences in the abundance of monocytes in general in these different mice ( Supplemental Fig. 1b ). Thus, together, these observations indicated that the CX3CR1 antibody used is indeed functional. To verify our findings across tissues, we performed the same experiments using splenocytes. As in the peripheral blood, an inverse correlation of Ly6C and CX3CR1 expression was only observed with the CX3CR1 reporter signal, but not using the CX3CR1 antibody (Z8-50, PE, BD Bioscience) (Fig. 3 a, b). In addition, as in the peripheral blood, we were able to proof the validity of the CX3CR1 antibody staining in different Cx3cr1 haplotypes (Fig. 3 c, d). In order to exclude fluorophore- or clone-specific effects, we additionally used another four commercially available and validated CX3CR1 antibodies. However, with none of them, we were able to reproduce the inverse correlation of Ly6C and CX3CR1 expression that we observed when using the reporter signal in Cx3cr1 GFP/+ mice ( Supplemental Fig. 2, 3 ). For further validation, we also used a second Cx3cr1 reporter mouse line in which a tamoxifen-dependent Cre recombinase controlled by the Cx3cr1 locus induces the expression of tdTomato (Cx3cr1 creER R26-tdTomato). Again, in the spleen of these mice, we observed Ly6C high CX3CR1 tdTomato − low and Ly6C low CX3CR1 tdTomato − high monocyte populations only with the CX3CR1 reporter signal, but not with antibody staining, fully supporting our findings in the Cx3cr1 GFP reporter mouse model ( Supplemental Fig. 4 ). Taken together, these data show that there is a discrepancy between CX3CR1 expression as determined by surface staining with validated CX3CR1 antibodies and quantification of fluorescent reporter signal in non-classical monocytes in mice. mRNA levels of Cx3cr1 and GFP are increased in Ly6C low compared to Ly6C high monocytes Finally, to better understand the reason underlying this discrepancy, we explored the expression of Cx3cr1 and GFP mRNA in monocytes from Cx3cr1 GFP/+ mice. Therefore, we sorted Cd11b + Ly6G − Cd115 + Ly6C high classical monocytes and Cd11b + Ly6G − Cd115 + Ly6C low non-classical monocytes from spleen and peripheral blood and performed qPCR analyses. The mRNA levels of Cx3cr1 as well as of GFP were substantially higher in non-classical compared to classical monocytes in blood and spleen (Fig. 4 ). In conclusion, these data suggest that differential CX3CR1 expression in non-classical and classical monocytes in mice is lost on the surface of these cells. Discussion As monocytes and downstream macrophages and DC are essential players of the immune system and are involved in different diseases, they are potential candidates for promising therapeutic approaches [ 30 – 32 ] . In rheumatoid arthritis, classical monocytes are shown to prevent arthritis and in contrast, deletion of non-classical monocytes was shown to prevent mice from developing arthritis [ 33 ] . Furthermore, Butovsky et al. showed in a mouse model of Amythotrophic lateral sclerosis (ALS) that the recruitment of inflammatory monocytes to the spinal cord plays an important role in disease progression [ 27 ] . Several therapeutics are already known to affect monocytes, but therapeutic approaches or interventions that specifically target monocytes are not available. Infliximab, a chimeric anti-tumour necrosis factor (TNF) antibody, was shown to induce monocyte apoptosis which could explain its powerful properties in patients with chronic active Crohn’s disease [ 34 ] . Hence, the accurate phenotyping of monocyte subsets is essential to derive meaningful conclusions from experimental models as well as to translate the insights into human disease. In this study, we challenge the current standard definition of mouse classical and non-classical monocytes as Ly6C high CX3CR1 low and Ly6C low CX3CR1 high , respectively. Indeed, we show that CX3CR1 is not differently expressed on the cell surface of classical and non-classical monocytes in mouse peripheral blood and spleen. This discrepancy can be explained by the fact that the initial landmark studies identifying different monocyte subsets used reporter mouse models and not antibody-based surface staining [ 15 ] to assess CX3CR1 expression. There are several genetically modified mice that have been used to investigate monocyte migration and trafficking including the knock-in/knock-out Cx3cr1 GFP reporter strain, in which the Cx3cr1 gene is replaced by a GFP reporter gene [ 15 , 23 ] . Therefore, heterozygous Cx3cr1 +/GFP mice with GFP substitution in only one Cx3cr1 allele can be used to identify Cx3cr1 -expressing monocytes whereas homozygous Cx3cr1 GFP/GFP mice show a CX3CR1 knock-out phenotype. In many studies, these mice were used and differential surface expression of CX3CR1 was assumed based on the differences in GFP expression [ 15 , 38 ] . However, using five different anti- CX3CR1 antibodies and two different reporter mouse models, we show that this is actually not the case and the CX3CR1 reporter signals do not match the actual surface expression of CX3CR1 as detected by anti- CX3CR1 antibodies. Interestingly, on mRNA level, Cx3cr1 expression was higher in non-classical compared to classical monocytes. Thus, it seems that CX3CR1 reporter mouse models correctly capture differential Cx3cr1 expression on the transcription level, while there is probably a different processing of GFP and CX3CR1 at the translation or post-translational level that maintains differences for GFP expression that are lost in the case of surface CX3CR1. Additional studies are needed to uncover the precise underlying mechanism and to determine whether e.g. mRNA stability [ 39 ] , post-translational mechanisms, [ 40 ] or transport and incorporation of the protein at the cell surface are altered [ 41 , 42 ] . In general, it is unknown to what extent the transcript levels by themselves dictate/predict cellular protein levels [ 43 – 45 ] and there are several examples for discrepant expression [ 46 – 50 ] . Taquet et al. showed a significant increase in somatostatin receptor 5 (SSR5) mRNA expression in Crohn’s disease patients however there was no increase in protein expression detected by immunohistochemistry and flow cytometry [ 51 ] . In synovial fibroblasts of rheumatoid arthritis patients masparin, a proteinase inhibitor with tumour suppressive functions, was intensively expressed on mRNA level but only slightly at the protein level [ 52 ] . It is well known that the stability of mRNAs depends on their nucleotide sequence affecting the secondary and tertiary structure of the mRNAs and thus the accessibility of various proteins to bind [ 39 ] . Furthermore, several mechanisms are known to have an impact on the expression level of a protein including translation rates, translation rate modulation, modulation of a protein’s half-life, protein synthesis delay and protein transport [ 43 ] . Beyond these open questions, the consequence of our findings is very clear: While we confirm that CX3CR1 reporter mouse models are a valuable tool to identify classical and non-classical monocytes, CX3CR1 antibody-based surface staining should not be used for this purpose. The discrepancy of flow cytometry gating based on reporter signal and antibody staining, as well as the discrepancy between mRNA and protein levels, should be carefully considered when investigating monocyte subsets and their functional role based on CX3CR1. Methods Mice All animals used in this study were housed in individually ventilated cages with a regular 12-hour day-night cycle and had free access to food and water at all times. Cx3cr1 GFP mice (B6.129P2(Cg)-Cx3cr1tm1Litt/J) were received from the Jackson Laboratory and were bred in-house to C57Bl/6J mice to obtain heterozygous Cx3cr1 +/GFP littermates. To receive all different haplotypes (homozygous GFP/GFP, heterozygous GFP/+ as well as wildtype +/+) heterozygous Cx3cr1 GFP/+ mice were crossed. Age and sex-matched C57Bl/6J wildtype mice were bred inhouse. Cx3cr1 creER R26-tdTomato mice (B6.Cx3cr1 tm2.1(cre/ERT2)Jung Gt(ROSA)26Sor tm9(CAG−tdTomato)Hze ) were available in-house. In order to induce the tdTomato reporter signal, mice were fed tamoxifen-containing food for 4 days before analysis. All mice were used for experiments according to approval by the Government of Lower Franconia after approval by the Animal Welfare Committee and all methods were performed according to relevant guidelines and all relevant ethical regulations. Mice were sacrificed by cervical dislocation and all procedures complied with ARRIVE guidelines. Isolation of cells Peripheral blood was collected from the facial vein. For erythrocyte removal, 2 ml of 1x BD Pharm Lyse™ lysing solution (BD Bioscience) was added to 70–80 µl of whole blood, vortexed and incubated for 15 minutes at room temperature (RT). Cells were washed two times with FACS buffer (phosphate buffered saline (PBS) supplemented with 1% fetal calf serum (FCS, PAN Biotech) and 2 mM EDTA) and were further processed for flow cytometry. Splenocytes were isolated as previously described [ 53 ] . In short, freshly isolated spleens were mashed through a 40 µm cell strainer and resuspended in 3 ml of ammonium-chloride-potassium lysis buffer (155 mM ammonium chloride; 19 mM potassium hydrogen carbonate and 0.68 mM EDTA; pH 7.27). After 3 minutes, the cells were washed with PBS and counted with a Neubauer counting chamber. For further flow cytometry analysis, 1–2 million splenocytes per sample were used. Flow cytometry and Fluorescence-activated cell sorting (FACS) Peripheral blood cells and splenocytes were stained for viable cells using the eBioscience Viability dye eFluor 506 or eFluor 780 (Invitrogen) for 30 minutes at 4°C and unspecific binding was blocked using the Fc Blocking Reagent (Miltenyi) according to the manufacturer’s protocol. Low-binding FACS tubes (Polypropylene round bottom Tube, FALCON) were used throughout. Cell surface staining was performed for 15 minutes at 4°C using the antibodies listed in Supplemental Table 1 . Fluorescently labelled cells were then fixed with 250 µl FluoroFix (BioLegend) for 1 hour at RT, washed two times with FACS buffer and analysed on a LSR Fortessa (BD Bioscience) instrument and with FlowJo™ v10.8 Software (BD Bioscience). For FACS, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from the heart. Therefore, whole blood was diluted at least 1:2 in PBS and 2 ml of Lympholyte cell separation media (Cedarlane) was slowly layered under the cell suspension and centrifuged for 20 minutes at 771 x g without break. Cells from the interphase were carefully removed and transferred into a new tube for washing. After counting, cells were stained for flow cytometry as mentioned above using the following antibodies listed in Supplemental Table 2 . Cd11b + Ly6G − Cd115 + Ly6C high and Cd11b + Ly6G − Cd115 + Ly6 low cells were sorted on an Astrios EQ Sorter (Beckman Coulter). RNA Isolation and quantitative polymerase chain reaction (qPCR) analysis RNA from sorted Cd11b + Ly6G − Cd115 + Ly6C high and Cd11b + Ly6G − Cd115 + Ly6 low cells was isolated using TRIzol (AMBION) according to the manufacturer´s protocol. Briefly, cells were either directly sorted into or resuspended in 500 µl of Trizol reagent, vortexed and frozen at -80°C. After thawing, 100 µl of chloroform was added and samples were centrifuged at 20,000 x g for 15 minutes at 4°C without brake to separate the protein, DNA and RNA components. The upper aqueous phase containing the RNA was carefully removed and 10 µg of glycogen (Thermo Fisher Scientific) was added. Subsequently, 250 µl of isopropanol (Carl Roth) was added and samples were incubated for 20 minutes on ice with regular vortexing intervals. Subsequently, the pellet was washed two times with 1 ml of 75% ethanol (Carl Roth) and dried at 37°C to remove remaining ethanol. The RNA pellet was then resuspended in 20 µl of RNase free water and incubated for further 10 minutes at 37°C. The concentration and purity of the extracted RNA was measured using a Nanodrop 2,000 spectrophotometer (Thermo Fisher Scientific). Total RNA was transcribed into complementary DNA (cDNA) using the AffinityScript Kit (Agilent). In short, 50 µg of mRNA were supplemented with poly d(T) primers and random primer mix and samples were incubated at 65°C for 5 minutes at 300 rounds per minute (rpm). After incubation for 10 minutes at RT, reverse transcriptase, 10x Affinity script buffer, dithiothreitol (DTT), desoxyribonucleotide triphosphate (dNTP) mixture and RNase-free water were added. The reverse transcription was performed for 1 hour at 42°C and 300 rpm followed by the inactivation of the enzyme at 70°C for 15 minutes. Primers for Cx3cr1 , enhanced-GFP ( E-GFP ) and HPRT as housekeeping gene were all purchased from Qiagen. qPCR analysis was run in duplicates using SybrSelect MasterMix (Thermo Fisher Scientific) and Quantitect Primer Assay (Qiagen). Duplicate values, in which the cycle threshold (Ct) value differed by more than one were excluded from further analyses. Statistics All statistical analyses were performed using GraphPad Prism software 9.5.1. Normality was tested using the Shapiro-Wilk test. If the samples were normally distributed, a paired t-Test was used. In contrast, if the samples were not normally distributed, a Wilcoxon Test was performed. Error bars in all graphs display the standard error of the mean (SEM). An α-value of p < 0.05 was defined as statistically significant. Significance levels are indicated by asterisks (* p < 0.05). Declarations Conflict of interest The author(s) declare no competing interests. Acknowledgment The authors thank J. Derdau, D. Dziony, S. Hofmann, J. Marcks and J. Schuster for their excellent technical assistance. In addition, the authors would like to thank the Cell Sorting Core Unit of the Friedrich-Alexander Universität Erlangen-Nürnberg for expert technical assistance. We acknowledge financial support by the German Research Foundation (ZU 377/4-1), Else Kröner-Fresenius-Stiftung (2021_CS.23) and Friedrich-Alexander-Universität Erlangen-Nürnberg within the funding programme “Open Access Publication Funding”. Author contributions KS and HG performed the experiments. KS and SZ designed the research. HG, MW, TMM, IA, GK, MFN and SZ provided protocols, reagents or designed experiments. KS, MW, MFN and SZ analysed and interpreted the data. KS and SZ drafted the manuscript; all authors critically read and revised the manuscript for important intellectual content and approved the final version. Data availability statement The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. All data generated or analysed during this study are included in this published article (and its Supplemental Information files). References Kawamura, S. & Ohteki, T. Monopoiesis in humans and mice. International Immunology 30, 503–509, doi: 10.1093/intimm/dxy063 (2018). Italiani, P. & Boraschi, D. From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation. Front Immunol 5, 514, doi: 10.3389/fimmu.2014.00514 (2014). 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Neurol Res 28, 787–793, doi: 10.1179/016164106x110364 (2006). Sarro, S. M. et al. Quantification of CD20 mRNA and protein levels in chronic lymphocytic leukemia suggests a post-transcriptional defect. Leuk Res 34, 1670–1673, doi: 10.1016/j.leukres.2010.06.031 (2010). Lichtinghagen, R. et al. Different mRNA and protein expression of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 in benign and malignant prostate tissue. Eur Urol 42, 398–406, doi: 10.1016/s0302-2838(02)00324-x (2002). Shebl, F. M. et al. Comparison of mRNA and protein measures of cytokines following vaccination with human papillomavirus-16 L1 virus-like particles. Cancer Epidemiol Biomarkers Prev 19, 978–981, doi: 10.1158/1055-9965.Epi-10-0064 (2010). Taquet, N. et al. Differential between protein and mRNA expression of CCR7 and SSTR5 receptors in Crohn's disease patients. Mediators Inflamm 2009, 285812, doi: 10.1155/2009/285812 (2009). Schedel, J. et al. Discrepancy between mRNA and protein expression of tumour suppressor maspin in synovial tissue may contribute to synovial hyperplasia in rheumatoid arthritis. Ann Rheum Dis 63, 1205–1211, doi: 10.1136/ard.2003.006312 (2004). Heidbreder, K. et al. Nr4a1-dependent non-classical monocytes are important for macrophage-mediated wound healing in the large intestine. Front Immunol 13, 1040775, doi: 10.3389/fimmu.2022.1040775 (2022). Additional Declarations No competing interests reported. Supplementary Files SupplementalFiles.pdf Cite Share Download PDF Status: Posted Version 1 posted 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 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-3262595","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":230730759,"identity":"d8c8c434-67a1-4ef4-95f9-d35b89dd1631","order_by":0,"name":"Katrin Sommer","email":"","orcid":"","institution":"University Hospital Erlangen and Friedrich-Alexander- Universität Erlangen-Nürnberg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Katrin","middleName":"","lastName":"Sommer","suffix":""},{"id":230730760,"identity":"492c0922-5c59-4830-9aa8-029777a98f08","order_by":1,"name":"Hilal Garibagaoglu","email":"","orcid":"","institution":"University Hospital Erlangen and Friedrich-Alexander- Universität Erlangen-Nürnberg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hilal","middleName":"","lastName":"Garibagaoglu","suffix":""},{"id":230730761,"identity":"410ce247-2cb0-4f60-9167-688094e27efa","order_by":2,"name":"Maximilian Wiendl","email":"","orcid":"","institution":"University Hospital Erlangen and Friedrich-Alexander- Universität Erlangen-Nürnberg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maximilian","middleName":"","lastName":"Wiendl","suffix":""},{"id":230730762,"identity":"fc65bce5-320d-43c9-8c62-d68b766c4594","order_by":3,"name":"Tanja M. Müller","email":"","orcid":"","institution":"University Hospital Erlangen and Friedrich-Alexander- Universität Erlangen-Nürnberg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"M.","lastName":"Müller","suffix":""},{"id":230730763,"identity":"94e9fe15-3864-44c5-b9c9-bb04c50eb49e","order_by":4,"name":"Imke Atreya","email":"","orcid":"","institution":"University Hospital Erlangen and Friedrich-Alexander- Universität Erlangen-Nürnberg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Imke","middleName":"","lastName":"Atreya","suffix":""},{"id":230730764,"identity":"29906490-b68c-4f13-affb-c780da2b002e","order_by":5,"name":"Gerhard Krönke","email":"","orcid":"","institution":"Charité - Universitätsmedizin Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gerhard","middleName":"","lastName":"Krönke","suffix":""},{"id":230730765,"identity":"c0133372-ddb3-46d0-ab8c-5939bcfbc3ba","order_by":6,"name":"Markus F. Neurath","email":"","orcid":"","institution":"University Hospital Erlangen and Friedrich-Alexander- Universität Erlangen-Nürnberg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Markus","middleName":"F.","lastName":"Neurath","suffix":""},{"id":230730766,"identity":"73079221-a19f-45a6-aac3-59f216327e08","order_by":7,"name":"Sebastian Zundler","email":"data:image/png;base64,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","orcid":"","institution":"University Hospital Erlangen and Friedrich-Alexander- Universität Erlangen-Nürnberg","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Zundler","suffix":""}],"badges":[],"createdAt":"2023-08-14 11:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3262595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3262595/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42775591,"identity":"1a7a6b9f-7661-4585-8f57-26f9fbcb08e4","added_by":"auto","created_at":"2023-09-07 14:28:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4023514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCX3CR1 antibody staining does not sufficiently discriminate mouse classical against non-classical monocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Representative gating strategy to identify monocytes in C57Bl/6 (WT) peripheral blood and splenocytes. Following exclusion of detritus based on SSC and FSC, we excluded duplicates and gated on viable cells. Monocytes were further defined as Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003e and Cd115\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Ly6C (HK1.1, BioLegend) vs. CX3CR1 (QA16A03, Biolegend) expression on Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003e peripheral monocytes and representative gating for Ly6C\u003csup\u003ehigh\u003c/sup\u003e and Ly6C\u003csup\u003elow\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Ly6C (HK1.1, BioLegend) vs. CX3CR1 (QA16A03, Biolegend) expression on Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003e monocytes from the spleen and representative gating for Ly6C\u003csup\u003ehigh\u003c/sup\u003e and Ly6C\u003csup\u003elow\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3262595/v1/8722c46e352c570eca5f2c30.png"},{"id":42775589,"identity":"88c888ad-13bc-4f21-a7a4-f244504a3504","added_by":"auto","created_at":"2023-09-07 14:28:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1842635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential CX3CR1 reporter fluorescence signal, but not antibody staining on peripheral blood monocytes in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Representative flow cytometry of peripheral blood monocytes from \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGRP/+\u003c/sup\u003e reporter mice. Monocytes were gated as described in \u003cem\u003eFigure 1\u003c/em\u003e. Left panel: CX3CR1-GFP fluorescence signal. Right panel: staining of the same reporter mice using the CX3CR1 (Z8-50, PE) antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Dot plots visualising the heatmap statistics of Ly6C on monocytes from \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e reporter mice. Red indicates a high expression of Ly6C while blue indicates low expression of Ly6C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Representative flow cytometry showing the staining of CX3CR1 on peripheral blood monocytes using the CX3CR1 (Z8-50, PE) antibody in C57Bl/6 WT (left panel) mice, WT littermates (\u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, middle left panel), heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice (right middle panel) and homozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/GFP\u003c/sup\u003e mice (right panel).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e Representative histograms showing the CX3CR1-GFP and CX3CR1 (Z8-50, PE) antibody signal in C57Bl/6 (WT, yellow) mice, WT littermates (\u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, blue), heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e (red) and homozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/GFP\u003c/sup\u003e mice (green) as well as the isotype control (grey). \u0026nbsp;Data are representative of at least three independent experiments.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3262595/v1/31c80435a8d81b5cf2ab390f.png"},{"id":42777238,"identity":"74976115-1a12-42a4-b39c-6663af45e6b3","added_by":"auto","created_at":"2023-09-07 14:36:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1898044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential CX3CR1 reporter fluorescence signal, but not antibody staining on splenic monocytes in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Representative flow cytometry of the GFP (left) and CX3CR1 antibody (Z8-50, PE, left) signals in monocytes of the spleen from \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e reporter mice, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Heatmap statistics of the Ly6C surface expression on monocytes from \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+ \u003c/sup\u003ereporter mice. Red indicates a high expression of Ly6C while blue indicates low expression of Ly6C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) \u003c/strong\u003eRepresentative flow cytometry showing the CX3CR1 (Z8-50, PE) antibody staining on splenic monocytes of C57Bl/6 WT (left panel) mice, WT littermates (\u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, middle left panel), heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice (right middle panel) and homozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/GFP\u003c/sup\u003e mice (right panel).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e Representative histograms showing the CX3CR1-GFP and CX3CR1 (Z8-50, PE) antibody signal in C57Bl/6 (WT, yellow) mice, WT littermates (\u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, blue), heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice (red) and homozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/GFP\u003c/sup\u003e mice (green) as well as the isotype control (grey). Data are representative of at least three independent experiments.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3262595/v1/14c292ad4c8033ba75d7dc4c.png"},{"id":42773518,"identity":"ec3ef955-653f-4ca4-9fe8-9bc227d9c81a","added_by":"auto","created_at":"2023-09-07 14:20:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":571170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCx3cr1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGFP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression on mRNA level in monocytes from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCx3cr1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eGFP/+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Quantitative qPCR of \u003cem\u003eCx3cr1\u003c/em\u003e mRNA (left panel, p = 0.024) and \u003cem\u003eenhanced-GFP (E-GFP)\u003c/em\u003e mRNA (right panel, p = 0.0144) expression relative to \u003cem\u003eHPRT\u003c/em\u003e in sorted classical (Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehigh\u003c/sup\u003e) and non-classical monocytes (Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elow\u003c/sup\u003e) from the peripheral blood (n = 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Quantitative qPCR of \u003cem\u003eCx3cr1\u003c/em\u003e mRNA (left panel, p = 0.015) and \u003cem\u003eenhanced-GFP (E-GFP)\u003c/em\u003e mRNA (right panel, p = 0.0167) expression in classical and non-classical monocytes from the spleen (n = 8).\u003c/p\u003e\n\u003cp\u003eFor normally distributed data the unpaired t-Test was used; for not normally distributed samples the Wilcoxon-Test was applied. CLM, classical monocytes; NCM, non-classical monocytes\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3262595/v1/217ee7b43c4beff43894c501.png"},{"id":45699961,"identity":"e6745737-7cec-4e55-bc30-3f2bc5a23628","added_by":"auto","created_at":"2023-11-02 03:37:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1558473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3262595/v1/31cb8742-8bd6-4041-b8cb-f4ad5f7e2d3b.pdf"},{"id":42773522,"identity":"bb0c894d-95bb-46a6-af37-09e7adefe422","added_by":"auto","created_at":"2023-09-07 14:20:38","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1156101,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3262595/v1/544c7c08f0cb7431f6236c01.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMonocytes are widely conserved cells of the myeloid linage. In the peripheral blood of mice, they account up to 4% of all leucocytes \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e and develop from hematopoietic stem cells (HSCs) in the bone marrow via the common myeloid progenitor \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. They are recruited from the bloodstream to surrounding tissue where they can differentiate into different macrophages or dendritic cells (DC) dependent on the tissue environment \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMonocytes are known to play a central role in both innate and adaptive immunity.\u003c/p\u003e \u003cp\u003eThey support and maintain tissue homeostasis by promoting the immune tolerance, contribute to the anti-microbial defence and are essential players of tissue repair and wound healing \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. However, monocytes are often considered as double-edged sword as they also contribute to the pathogenesis and progression of chronic inflammatory conditions like inflammatory bowel disease, rheumatoid arthritis or multiple sclerosis \u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOver time, our understanding of monocytes has evolved from viewing them as a homogeneous population simply being macrophage precursors to a heterogeneous population with various functions \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The first evidence of different monocyte subsets was provided by Geissmann et al. identifying a CX3CR1\u003csup\u003ehigh\u003c/sup\u003e and a CX3CR1\u003csup\u003elow\u003c/sup\u003e subset with different phenotypic and functional properties, which were confirmed in later reports \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. CX3CR1 is a 7-transmembrane receptor coupled to heterotrimeric G proteins that is important for the adhesion of leukocytes, cell survival and the recruitment of immune cell subpopulations \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Several studies demonstrated that CX3CR1 signalling is an essential survival factor of monocytes \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Importantly, these initial landmark studies were based on the use of reporter mouse models. Subsequent studies furthermore identified Ly6C as specific marker for discriminating monocytes in two phenotypically and functionally different subtypes \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Conventionally, driven by these studies, mouse monocytes are divided into a classical and a non-classical subset based on their inverse correlation of CX3CR1 and Ly6C surface expression.\u003c/p\u003e \u003cp\u003eClassical monocytes, also called inflammatory monocytes, are defined as Ly6C\u003csup\u003ehigh\u003c/sup\u003e and CX3CR1\u003csup\u003elow\u003c/sup\u003e and are recruited to sites of inflammation at higher rates, where they recognize and phagocytose pathogens and are able to attract other immune cells by secreting cytokines and antimicrobial factors. On the other hand, non-classical monocytes, defined as Ly6C\u003csup\u003elow\u003c/sup\u003e CX3CR1\u003csup\u003ehigh\u003c/sup\u003e, are characterized by their ability to patrol along the vascular endothelium, to remove cell debris and to promote tissue repair \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to reproduce and validate conventional monocyte subtyping in WT and reporter mice. Therefore, we used \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP\u003c/sup\u003e reporter mice, in which the \u003cem\u003eCx3cr1\u003c/em\u003e gene is replaced by a green fluorescent protein (GFP) reporter gene. Heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/GFP\u003c/sup\u003e mice with \u003cem\u003eGFP\u003c/em\u003e substitution in only one \u003cem\u003eCx3cr1\u003c/em\u003e allele were used as reporters to identify \u003cem\u003eCx3cr1\u003c/em\u003e-expressing monocytes whereas homozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/GFP\u003c/sup\u003e mice show a functional CX3CR1 knock-out \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Comparing reporter signal and several validated fluorochrome-coupled antibodies head-to-head, we show for the first time that there is a discrepancy between the CX3CR1 reporter gene signal and CX3CR1 surface expression as detected by flow cytometry. However, on mRNA levels, we observed indeed a higher expression of \u003cem\u003eCx3cr1\u003c/em\u003e in non-classical as compared to classical monocytes in peripheral blood and spleen. These data suggest that CX3CR1 antibody surface staining should be used with caution to profile classical and non-classical monocytes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eMonocytes from the peripheral blood and spleen of C57Bl/6 mice show no inverse correlation of Ly6C and CX3CR1 expression by flow cytometry\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn a first step, we aimed to set up a flow cytometry panel to characterise classical and non-classical monocytes based on Ly6C and CX3CR1 as suggested in the literature \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Thus, we isolated cells from the peripheral blood of C57Bl/6 mice and analysed them by flow cytometry. Monocytes were defined as Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In addition, these cells were also Cd172a\u003csup\u003e+\u003c/sup\u003e and SiglecF\u003csup\u003e\u0026minus;\u003c/sup\u003e excluding contamination with type 1 conventional dendritic cells (which are Cd172a\u003csup\u003e\u0026minus;\u003c/sup\u003e) and neutrophils (which are SiglecF\u003csup\u003e+\u003c/sup\u003e) (\u003cem\u003eSupplemental Fig.\u0026nbsp;1a\u003c/em\u003e). In the next step, we used Ly6C (HK1.4, BioLegend) and CX3CR1 (QA16A03, BioLegend) to subcluster these monocytes. While there was a clear separation into Ly6C\u003csup\u003ehigh\u003c/sup\u003e and Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes, we did not observe any relevant differences in the CX3CR1 expression of these monocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Similar observations were made using splenocytes from C57Bl/6 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Collectively, these data put the inverse correlation of the surface expression of Ly6C and CX3CR1 on mouse monocytes into question.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDiscrepancy between fluorescent reporter and antibody-based assessment of CX3CR1 expression\u003c/h2\u003e \u003cp\u003eAs the initial studies identifying classical and non-classical monocytes using CX3CR1 were based on reporter mouse models \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, we also investigated the surface expression of CX3CR1 and Ly6C in the peripheral blood of \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP\u003c/sup\u003e mice by flow cytometry. Therefore, we used heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice, which express \u003cem\u003eCx3cr1\u003c/em\u003e on one allele and \u003cem\u003eGFP\u003c/em\u003e under the control of the \u003cem\u003eCx3cr1\u003c/em\u003e promoter on the other allele. First, we explored whether we can reproduce the inverse correlation of Ly6C and CX3CR1 expression on monocytes based on the CX3CR1 reporter signal. Indeed, we were able to distinguish a Ly6C\u003csup\u003ehigh\u003c/sup\u003eCX3CR1\u003csup\u003eGFP\u0026thinsp;\u0026minus;\u0026thinsp;low\u003c/sup\u003e and a Ly6C\u003csup\u003elow\u003c/sup\u003eCX3CR1\u003csup\u003eGFP\u0026thinsp;\u0026minus;\u0026thinsp;high\u003c/sup\u003e population in accordance to previous reports (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) \u003csup\u003e[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. However, using a CX3CR1 antibody (Z8-50, PE, BD Bioscience), we were not able to distinguish these populations on peripheral blood monocytes in the same \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Consistently, we did not observe any correlation between antibody-based CX3CR1 and Ly6C expression nor between CX3CR1 antibody and reporter signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, in a next step, we aimed to verify that the antibody used is indeed functional and able to detect CX3CR1. To this end, we took advantage of different haplotypes of the \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP\u003c/sup\u003e reporter mice. As expected, C57Bl/6 WT and \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice showed no expression of CX3CR1-GFP, but similar expression of CX3CR1 as detected by antibody staining on peripheral blood monocytes. In contrast, monocytes from heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice showed expression of CX3CR1-GFP as well as CX3CR1 antibody staining, which was less intense than on C57Bl/6 WT and \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e monocytes. Monocytes from homozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/GFP\u003c/sup\u003e mice also expressed CX3CR1-GFP (and more intense than \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e counterparts), but there was no CX3CR1 antibody staining as expected due to the functional knockout of CX3CR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). Importantly, we did not observe any differences in the abundance of monocytes in general in these different mice (\u003cem\u003eSupplemental Fig.\u0026nbsp;1b\u003c/em\u003e). Thus, together, these observations indicated that the CX3CR1 antibody used is indeed functional.\u003c/p\u003e \u003cp\u003eTo verify our findings across tissues, we performed the same experiments using splenocytes. As in the peripheral blood, an inverse correlation of Ly6C and CX3CR1 expression was only observed with the CX3CR1 reporter signal, but not using the CX3CR1 antibody (Z8-50, PE, BD Bioscience) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In addition, as in the peripheral blood, we were able to proof the validity of the CX3CR1 antibody staining in different \u003cem\u003eCx3cr1\u003c/em\u003e haplotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to exclude fluorophore- or clone-specific effects, we additionally used another four commercially available and validated CX3CR1 antibodies. However, with none of them, we were able to reproduce the inverse correlation of Ly6C and CX3CR1 expression that we observed when using the reporter signal in \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice (\u003cem\u003eSupplemental Fig.\u0026nbsp;2, 3\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFor further validation, we also used a second \u003cem\u003eCx3cr1\u003c/em\u003e reporter mouse line in which a tamoxifen-dependent Cre recombinase controlled by the \u003cem\u003eCx3cr1\u003c/em\u003e locus induces the expression of tdTomato (Cx3cr1\u003csup\u003ecreER\u003c/sup\u003e R26-tdTomato). Again, in the spleen of these mice, we observed Ly6C\u003csup\u003ehigh\u003c/sup\u003eCX3CR1\u003csup\u003etdTomato\u0026thinsp;\u0026minus;\u0026thinsp;low\u003c/sup\u003e and Ly6C\u003csup\u003elow\u003c/sup\u003eCX3CR1\u003csup\u003etdTomato\u0026thinsp;\u0026minus;\u0026thinsp;high\u003c/sup\u003e monocyte populations only with the CX3CR1 reporter signal, but not with antibody staining, fully supporting our findings in the \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP\u003c/sup\u003e reporter mouse model (\u003cem\u003eSupplemental Fig.\u0026nbsp;4\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eTaken together, these data show that there is a discrepancy between CX3CR1 expression as determined by surface staining with validated CX3CR1 antibodies and quantification of fluorescent reporter signal in non-classical monocytes in mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003emRNA levels of Cx3cr1 and GFP are increased in Ly6C\u003csup\u003elow\u003c/sup\u003e compared to Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes\u003c/h2\u003e \u003cp\u003eFinally, to better understand the reason underlying this discrepancy, we explored the expression of \u003cem\u003eCx3cr1\u003c/em\u003e and \u003cem\u003eGFP\u003c/em\u003e mRNA in monocytes from \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice. Therefore, we sorted Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehigh\u003c/sup\u003e classical monocytes and Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elow\u003c/sup\u003e non-classical monocytes from spleen and peripheral blood and performed qPCR analyses. The mRNA levels of \u003cem\u003eCx3cr1\u003c/em\u003e as well as of \u003cem\u003eGFP\u003c/em\u003e were substantially higher in non-classical compared to classical monocytes in blood and spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In conclusion, these data suggest that differential CX3CR1 expression in non-classical and classical monocytes in mice is lost on the surface of these cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs monocytes and downstream macrophages and DC are essential players of the immune system and are involved in different diseases, they are potential candidates for promising therapeutic approaches \u003csup\u003e[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn rheumatoid arthritis, classical monocytes are shown to prevent arthritis and in contrast, deletion of non-classical monocytes was shown to prevent mice from developing arthritis \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Furthermore, Butovsky et al. showed in a mouse model of Amythotrophic lateral sclerosis (ALS) that the recruitment of inflammatory monocytes to the spinal cord plays an important role in disease progression \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Several therapeutics are already known to affect monocytes, but therapeutic approaches or interventions that specifically target monocytes are not available. Infliximab, a chimeric anti-tumour necrosis factor (TNF) antibody, was shown to induce monocyte apoptosis which could explain its powerful properties in patients with chronic active Crohn\u0026rsquo;s disease \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Hence, the accurate phenotyping of monocyte subsets is essential to derive meaningful conclusions from experimental models as well as to translate the insights into human disease.\u003c/p\u003e \u003cp\u003eIn this study, we challenge the current standard definition of mouse classical and non-classical monocytes as Ly6C\u003csup\u003ehigh\u003c/sup\u003eCX3CR1\u003csup\u003elow\u003c/sup\u003e and Ly6C\u003csup\u003elow\u003c/sup\u003eCX3CR1\u003csup\u003ehigh\u003c/sup\u003e, respectively. Indeed, we show that CX3CR1 is not differently expressed on the cell surface of classical and non-classical monocytes in mouse peripheral blood and spleen. This discrepancy can be explained by the fact that the initial landmark studies identifying different monocyte subsets used reporter mouse models and not antibody-based surface staining \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e to assess CX3CR1 expression. There are several genetically modified mice that have been used to investigate monocyte migration and trafficking including the knock-in/knock-out \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP\u003c/sup\u003e reporter strain, in which the \u003cem\u003eCx3cr1\u003c/em\u003e gene is replaced by a \u003cem\u003eGFP\u003c/em\u003e reporter gene \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Therefore, heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/GFP\u003c/sup\u003e mice with \u003cem\u003eGFP\u003c/em\u003e substitution in only one \u003cem\u003eCx3cr1\u003c/em\u003e allele can be used to identify \u003cem\u003eCx3cr1\u003c/em\u003e-expressing monocytes whereas homozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/GFP\u003c/sup\u003e mice show a CX3CR1 knock-out phenotype. In many studies, these mice were used and differential surface expression of CX3CR1 was assumed based on the differences in GFP expression \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. However, using five different anti- CX3CR1 antibodies and two different reporter mouse models, we show that this is actually not the case and the CX3CR1 reporter signals do not match the actual surface expression of CX3CR1 as detected by anti- CX3CR1 antibodies. Interestingly, on mRNA level, \u003cem\u003eCx3cr1\u003c/em\u003e expression was higher in non-classical compared to classical monocytes. Thus, it seems that CX3CR1 reporter mouse models correctly capture differential \u003cem\u003eCx3cr1\u003c/em\u003e expression on the transcription level, while there is probably a different processing of GFP and CX3CR1 at the translation or post-translational level that maintains differences for GFP expression that are lost in the case of surface CX3CR1. Additional studies are needed to uncover the precise underlying mechanism and to determine whether e.g. mRNA stability \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, post-translational mechanisms, \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e or transport and incorporation of the protein at the cell surface are altered \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn general, it is unknown to what extent the transcript levels by themselves dictate/predict cellular protein levels \u003csup\u003e[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e and there are several examples for discrepant expression \u003csup\u003e[\u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Taquet et al. showed a significant increase in somatostatin receptor 5 (SSR5) mRNA expression in Crohn\u0026rsquo;s disease patients however there was no increase in protein expression detected by immunohistochemistry and flow cytometry \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. In synovial fibroblasts of rheumatoid arthritis patients masparin, a proteinase inhibitor with tumour suppressive functions, was intensively expressed on mRNA level but only slightly at the protein level \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is well known that the stability of mRNAs depends on their nucleotide sequence affecting the secondary and tertiary structure of the mRNAs and thus the accessibility of various proteins to bind \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Furthermore, several mechanisms are known to have an impact on the expression level of a protein including translation rates, translation rate modulation, modulation of a protein\u0026rsquo;s half-life, protein synthesis delay and protein transport \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBeyond these open questions, the consequence of our findings is very clear: While we confirm that CX3CR1 reporter mouse models are a valuable tool to identify classical and non-classical monocytes, CX3CR1 antibody-based surface staining should not be used for this purpose. The discrepancy of flow cytometry gating based on reporter signal and antibody staining, as well as the discrepancy between mRNA and protein levels, should be carefully considered when investigating monocyte subsets and their functional role based on CX3CR1.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eAll animals used in this study were housed in individually ventilated cages with a regular 12-hour day-night cycle and had free access to food and water at all times. \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP\u003c/sup\u003e mice (B6.129P2(Cg)-Cx3cr1tm1Litt/J) were received from the Jackson Laboratory and were bred in-house to C57Bl/6J mice to obtain heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e+/GFP\u003c/sup\u003e littermates. To receive all different haplotypes (homozygous GFP/GFP, heterozygous GFP/+ as well as wildtype +/+) heterozygous \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP/+\u003c/sup\u003e mice were crossed. Age and sex-matched C57Bl/6J wildtype mice were bred inhouse. \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003ecreER\u003c/sup\u003e R26-tdTomato mice (B6.Cx3cr1\u003csup\u003etm2.1(cre/ERT2)Jung\u003c/sup\u003eGt(ROSA)26Sor\u003csup\u003etm9(CAG\u0026minus;tdTomato)Hze\u003c/sup\u003e) were available in-house. In order to induce the tdTomato reporter signal, mice were fed tamoxifen-containing food for 4 days before analysis. All mice were used for experiments according to approval by the Government of Lower Franconia after approval by the Animal Welfare Committee and all methods were performed according to relevant guidelines and all relevant ethical regulations. Mice were sacrificed by cervical dislocation and all procedures complied with ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of cells\u003c/h2\u003e \u003cp\u003ePeripheral blood was collected from the facial vein. For erythrocyte removal, 2 ml of 1x BD Pharm Lyse\u0026trade; lysing solution (BD Bioscience) was added to 70\u0026ndash;80 \u0026micro;l of whole blood, vortexed and incubated for 15 minutes at room temperature (RT). Cells were washed two times with FACS buffer (phosphate buffered saline (PBS) supplemented with 1% fetal calf serum (FCS, PAN Biotech) and 2 mM EDTA) and were further processed for flow cytometry.\u003c/p\u003e \u003cp\u003eSplenocytes were isolated as previously described \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. In short, freshly isolated spleens were mashed through a 40 \u0026micro;m cell strainer and resuspended in 3 ml of ammonium-chloride-potassium lysis buffer (155 mM ammonium chloride; 19 mM potassium hydrogen carbonate and 0.68 mM EDTA; pH 7.27). After 3 minutes, the cells were washed with PBS and counted with a Neubauer counting chamber. For further flow cytometry analysis, 1\u0026ndash;2\u0026nbsp;million splenocytes per sample were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry and Fluorescence-activated cell sorting (FACS)\u003c/h2\u003e \u003cp\u003ePeripheral blood cells and splenocytes were stained for viable cells using the eBioscience Viability dye eFluor 506 or eFluor 780 (Invitrogen) for 30 minutes at 4\u0026deg;C and unspecific binding was blocked using the Fc Blocking Reagent (Miltenyi) according to the manufacturer\u0026rsquo;s protocol. Low-binding FACS tubes (Polypropylene round bottom Tube, FALCON) were used throughout. Cell surface staining was performed for 15 minutes at 4\u0026deg;C using the antibodies listed in \u003cem\u003eSupplemental Table\u0026nbsp;1\u003c/em\u003e. Fluorescently labelled cells were then fixed with 250 \u0026micro;l FluoroFix (BioLegend) for 1 hour at RT, washed two times with FACS buffer and analysed on a LSR Fortessa (BD Bioscience) instrument and with FlowJo\u0026trade; v10.8 Software (BD Bioscience).\u003c/p\u003e \u003cp\u003eFor FACS, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from the heart. Therefore, whole blood was diluted at least 1:2 in PBS and 2 ml of Lympholyte cell separation media (Cedarlane) was slowly layered under the cell suspension and centrifuged for 20 minutes at 771 x g without break. Cells from the interphase were carefully removed and transferred into a new tube for washing. After counting, cells were stained for flow cytometry as mentioned above using the following antibodies listed in \u003cem\u003eSupplemental Table\u0026nbsp;2\u003c/em\u003e. Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehigh\u003c/sup\u003e and Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6\u003csup\u003elow\u003c/sup\u003e cells were sorted on an Astrios EQ Sorter (Beckman Coulter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA Isolation and quantitative polymerase chain reaction (qPCR) analysis\u003c/h2\u003e \u003cp\u003eRNA from sorted Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehigh\u003c/sup\u003e and Cd11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCd115\u003csup\u003e+\u003c/sup\u003eLy6\u003csup\u003elow\u003c/sup\u003e cells was isolated using TRIzol (AMBION) according to the manufacturer\u0026acute;s protocol. Briefly, cells were either directly sorted into or resuspended in 500 \u0026micro;l of Trizol reagent, vortexed and frozen at -80\u0026deg;C. After thawing, 100 \u0026micro;l of chloroform was added and samples were centrifuged at 20,000 x g for 15 minutes at 4\u0026deg;C without brake to separate the protein, DNA and RNA components. The upper aqueous phase containing the RNA was carefully removed and 10 \u0026micro;g of glycogen (Thermo Fisher Scientific) was added. Subsequently, 250 \u0026micro;l of isopropanol (Carl Roth) was added and samples were incubated for 20 minutes on ice with regular vortexing intervals. Subsequently, the pellet was washed two times with 1 ml of 75% ethanol (Carl Roth) and dried at 37\u0026deg;C to remove remaining ethanol. The RNA pellet was then resuspended in 20 \u0026micro;l of RNase free water and incubated for further 10 minutes at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe concentration and purity of the extracted RNA was measured using a Nanodrop 2,000 spectrophotometer (Thermo Fisher Scientific). Total RNA was transcribed into complementary DNA (cDNA) using the AffinityScript Kit (Agilent). In short, 50 \u0026micro;g of mRNA were supplemented with poly d(T) primers and random primer mix and samples were incubated at 65\u0026deg;C for 5 minutes at 300 rounds per minute (rpm). After incubation for 10 minutes at RT, reverse transcriptase, 10x Affinity script buffer, dithiothreitol (DTT), desoxyribonucleotide triphosphate (dNTP) mixture and RNase-free water were added. The reverse transcription was performed for 1 hour at 42\u0026deg;C and 300 rpm followed by the inactivation of the enzyme at 70\u0026deg;C for 15 minutes.\u003c/p\u003e \u003cp\u003ePrimers for \u003cem\u003eCx3cr1\u003c/em\u003e, \u003cem\u003eenhanced-GFP\u003c/em\u003e (\u003cem\u003eE-GFP\u003c/em\u003e) and \u003cem\u003eHPRT\u003c/em\u003e as housekeeping gene were all purchased from Qiagen. qPCR analysis was run in duplicates using SybrSelect MasterMix (Thermo Fisher Scientific) and Quantitect Primer Assay (Qiagen). Duplicate values, in which the cycle threshold (Ct) value differed by more than one were excluded from further analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism software 9.5.1. Normality was tested using the Shapiro-Wilk test. If the samples were normally distributed, a paired t-Test was used. In contrast, if the samples were not normally distributed, a Wilcoxon Test was performed. Error bars in all graphs display the standard error of the mean (SEM). An α-value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was defined as statistically significant. Significance levels are indicated by asterisks (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank J. Derdau, D. Dziony, S. Hofmann, J. Marcks and J. Schuster for their excellent technical assistance. In addition, the authors would like to thank the Cell Sorting Core Unit of the Friedrich-Alexander Universität Erlangen-Nürnberg for expert technical assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe acknowledge financial support by the German Research Foundation (ZU 377/4-1), Else Kröner-Fresenius-Stiftung (2021_CS.23) and Friedrich-Alexander-Universität Erlangen-Nürnberg within the funding programme “Open Access Publication Funding”.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKS and HG performed the experiments. KS and SZ designed the research. HG, MW, TMM, IA, GK, MFN and SZ provided protocols, reagents or designed experiments. KS, MW, MFN and SZ analysed and interpreted the data. KS and SZ drafted the manuscript; all authors critically read and revised the manuscript for important intellectual content and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article (and its Supplemental Information files).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKawamura, S. \u0026amp; Ohteki, T. Monopoiesis in humans and mice. 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Front Immunol 13, 1040775, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2022.1040775\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2022.1040775\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Classical monocytes, non-classical monocytes, C-X3-C Motif Chemokine Receptor 1 (CX3CR1), Lymphocyte antigen 6C2 (Ly6C)","lastPublishedDoi":"10.21203/rs.3.rs-3262595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3262595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMonocytes as well as downstream macrophages and dendritic cells are essential players of the immune system fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies in reporter models, mouse monocytes are divided into a classical and a non-classical subset based on their inversely correlating surface expression of Ly6C and CX3CR1.\u003c/p\u003e \u003cp\u003eHere, we analysed the expression of CX3CR1 by flow cytometry using several validated fluorochrome-coupled CX3CR1 antibodies and compared them with the reporter gene signal of a \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eGFP\u003c/sup\u003e reporter mouse strain as well as of tamoxifen-inducible \u003cem\u003eCx3cr1\u003c/em\u003e reporter mice.\u003c/p\u003e \u003cp\u003eAlthough we were able to validate the specificity of several fluorochrome-coupled CX3CR1 flow cytometry antibodies, mouse Ly6C\u003csup\u003ehigh\u003c/sup\u003e classical and Ly6C\u003csup\u003elow\u003c/sup\u003e non-classical monocytes showed no differences in CX3CR1 expression levels in peripheral blood and spleen, when stained with these antibodies. To the contrary, in reporter mice, we were able to reproduce the inverse correlation of CX3CR1 reporter gene signal and Ly6C surface expression. As determined by qPCR, the \u003cem\u003eCx3cr1\u003c/em\u003e mRNA expression correlated with the GFP-reporter gene expression as quantified by flow cytometry.\u003c/p\u003e \u003cp\u003eIn conclusion, our data suggest that there is differential transcription, but not surface expression of CX3CR1 between classical and non-classical monocytes, which limits the suitability of CX3CR1 for phenotyping monocyte subsets by antibody staining.\u003c/p\u003e","manuscriptTitle":"Discrepant phenotyping of monocytes based on CX3CR1 using fluorescent reporters and antibodies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-07 14:20:33","doi":"10.21203/rs.3.rs-3262595/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d1202475-20df-462f-ab11-f68cb7fd4e91","owner":[],"postedDate":"September 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":24417305,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages"},{"id":24417306,"name":"Biological sciences/Immunology/Chemokines"}],"tags":[],"updatedAt":"2023-11-02T03:29:36+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-07 14:20:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3262595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3262595","identity":"rs-3262595","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}