IMDM-20 enhances neutrophilic features during DMSO-mediated differentiation of HL-60 cells

IMDM-20 enhances neutrophilic features during DMSO-mediated differentiation of HL-60 cells | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 23 April 2025 V1 Latest version Share on IMDM-20 enhances neutrophilic features during DMSO-mediated differentiation of HL-60 cells Authors : Jorge Andrés Cázares-Preciado 0000-0003-2661-584X , José Antonio Cruz-Cardenas , Alejandra López-Arredondo , Marco Vinicio Gallardo-Camarena , and Marion E. G. Brunck [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174537723.38313669/v1 Published Biochemistry and Biophysics Reports Version of record Peer review timeline 376 views 167 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The promyelocytic HL-60 cell line can be differentiated toward neutrophil-like cells and has been historically used as a surrogate to study human neutrophil biology in vitro . Multiple differentiation protocols have been reported to generate neutrophil-like HL-60 cells, with limited consideration of how methodological variations might influence cell identity and functions. Here, we performed a systematic search of the research literature published between January 9 th 2020, and January 9 th 2025, to investigate the current heterogeneity in protocols used to differentiate HL-60 towards neutrophil-like cells. A total of 71 studies published in 5 years employed 41 distinct protocols. The 3 most prevalent conditions to maintain HL-60 cells were IMDM with 20% FBS (IMDM-20), DMEM with 10% FBS (DMEM-10), and RPMI-1640 with 10% FBS (RPMI-10). Over 90% of protocols applied 1-1.57% DMSO as differentiating agent to produce neutrophil-like cells. In the laboratory, we compared the 3 most common culture media applied during neutrophil-like cell differentiation with 1.3% DMSO over 5 and 7 days. Using IMDM-20 led to the highest proliferation rate and cell yield during differentiation. Neutrophil-like cells produced in IMDM-20 and RPMI-10 exhibited significantly higher proportions of CD15 + CD11b + cells, and significantly higher bacterial clearance compared to DMEM-10. Culture media did not affect phagocytosis, but using RPMI-10 over 5 days led to significantly higher ability to produce ROS. IMDM-20 produced significantly more IL-6 and IL-1β in culture supernatant following stimulation with immune complexes. Overall, the results support the use of IMDM-20 with 1.3% DMSO to differentiate HL-60 to study neutrophil biology in vitro . IMDM-20 enhances neutrophilic features during DMSO-mediated differentiation of HL-60 cells Jorge Andrés Cázares-Preciado 1 , José Antonio Cruz-Cardenas 1 , Alejandra López-Arredondo 1 , Marco Vinicio Gallardo-Camarena 1 , Marion E. G. Brunck 2* * Corresponding author : [email protected] ; [email protected] ORCID: Jorge Andrés Cázares-Preciado: 0003-2661-584X Alejandra López-Arredondo 0003-3557-5122 Marion E. G. Brunck ORCID: 0001-8994-8276 Affiliations: 1 Tecnológico de Monterrey, Escuela de Ingeniería y Ciencias, Av. Eugenio Garza Sada 2501 Sur, Tecnologico, 64849, Monterrey, Nuevo León, México 2 Tecnologico de Monterrey, Institute for Obesity Research, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849, Monterrey, Nuevo León, México Abstract The promyelocytic HL-60 cell line can be differentiated toward neutrophil-like cells and has been historically used as a surrogate to study human neutrophil biology in vitro . Multiple differentiation protocols have been reported to generate neutrophil-like HL-60 cells, with limited consideration of how methodological variations might influence cell identity and functions. Here, we performed a systematic search of the research literature published between January 9 th 2020, and January 9 th 2025, to investigate the current heterogeneity in protocols used to differentiate HL-60 towards neutrophil-like cells. A total of 71 studies published in 5 years employed 41 distinct protocols. The 3 most prevalent conditions to maintain HL-60 cells were IMDM with 20% FBS (IMDM-20), DMEM with 10% FBS (DMEM-10), and RPMI-1640 with 10% FBS (RPMI-10). Over 90% of protocols applied 1-1.57% DMSO as differentiating agent to produce neutrophil-like cells. In the laboratory, we compared the 3 most common culture media applied during neutrophil-like cell differentiation with 1.3% DMSO over 5 and 7 days. Using IMDM-20 led to the highest proliferation rate and cell yield during differentiation. Neutrophil-like cells produced in IMDM-20 and RPMI-10 exhibited significantly higher proportions of CD15 + CD11b + cells, and significantly higher bacterial clearance compared to DMEM-10. Culture media did not affect phagocytosis, but using RPMI-10 over 5 days led to significantly higher ability to produce ROS. IMDM-20 produced significantly more IL-6 and IL-1β in culture supernatant following stimulation with immune complexes. Overall, the results support the use of IMDM-20 with 1.3% DMSO to differentiate HL-60 to study neutrophil biology in vitro . Keywords HL-60, neutrophils, culture media, differentiation. Abbreviations DMSO: dimethyl sulfoxide MFI: median fluorescence intensity IC: immune complex ATRA: all-trans retinoic acid FBS: fetal bovine serum IMDM: Iscove’s modified Dulbecco’s Medium RPMI-1640: Roswell Park Memorial Institute DMEM: Dulbecco’s Modified Eagle Medium IMDM-20: Iscove’s modified Dulbecco’s Medium supplemented with 20% FBS RPMI-10: Roswell Park Memorial Institute supplemented with 10% FBS DMEM-10: Dulbecco’s Modified Eagle Medium supplemented with 10% FBS INTRODUCTION Neutrophils are the most abundant leukocytes in human blood. Their study ex vivo is hindered by spontaneous activation post-enrichment and by their short lifespan 1 . Researchers have used model cell lines to facilitate studies of human neutrophil biology 2 . The HL-60 cell line originated from a peripheral blood donation from an adult female patient presenting acute promyelocytic leukemia. The original article described a heterogeneous cell culture composed predominantly of promyelocytes and myeloblasts, with a minor representation of monocytes, bands, and segmented neutrophil-like cells 3 . The first report of HL-60-differentiated neutrophil-like cells described using 1.3% dimethyl sulfoxide (DMSO) in RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS) for 5–7 days 4 . Others have successfully differentiated HL-60 towards neutrophil-like cells using ATRA, cyclic AMP, or dimethylformamide 2 . The American Type Culture Collection (ATCC) recommends propagating HL-60 cells in Iscove’s modified Dulbecco’s Medium (IMDM) supplemented with 20% FBS (IMDM-20), without specifying media or differentiation agent to produce neutrophil-like cells 5 . In the literature, a range of media has been reported to propagate HL-60 cells. While some articles follow the ATCC’s recommendations 6 , others describe using RPMI-1640 supplemented with 10% FBS (RPMI-10), or Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (DMEM-10), often without providing a rationale for these choices 7,8 . For inducing differentiation using DMSO, various concentrations have been reported DMSO, with the length of this stimulation ranging from 1 9 to 9 days 10 . Herein, we address discrepancies in protocol choice to investigate HL-60-derived neutrophil-like cells. We performed a systematic search of the recent literature describing the production of HL-60-derived neutrophil-like cell between January 9, 2020 and January 9, 2025. After manual curation of search results, 75 studies were included in the analysis that overall applied 41 distinct protocols, highlighting heterogeneity and lack of consensus. In the laboratory, we employed the 3 most reported maintenance media, together with 1.3% DMSO to produce HL-60-derived neutrophil-like cells. Phagocytosis was not affected by culture media, but in vitro killing of E. coli was significantly impaired using DMEM. ROS production was enhanced when using RPMI in a 5-day differentiation, while using IMDM media lead to broader cytokines profile, more closely resembling the diverse secretion patterns of peripheral blood neutrophils. This study may help guide the selection of HL-60 culture protocols for in vitro biomedical research. RESULTS AND DISCUSSION Between 2021 and 2025, RPMI-10 was prevalently used as maintenance media for HL-60 cells. We performed a systematic search of the literature to identify current culture conditions for HL-60 and HL-60 neutrophil-like cells. Between 2021 and 2025, we identified a total of 89 articles from which 14 were excluded ( Supplementary Fig. 1, Supplementary table 1 ). A total of 75 studies were included in the analysis. Among those, 71 studies (94.6%) induced neutrophilic differentiation of HL-60 cells, while 4 articles described HL-60 cells in maintenance media to study neutrophil biology. Extracted metadata for each paper are available in Supplementary table 2. Forty-one distinct protocols were employed from 71 included studies. Most studies reported using RPMI (78.3% of included studies) followed by IMDM (14.8%) and DMEM (2.7%, Fig. 1a ) to culture HL-60 cells. Overall, the predominant FBS concentration used for media supplementation was 10% (72.2% of papers reporting culture conditions). In studies reporting using IMDM, the most common FBS concentration was 20%, in line with ATCC recommendations (63.6% of papers reporting using IMDM, Fig. 1b ). Most studies using RPMI reported 10% FBS, the most common concentration (79.3%, Fig. 1c ). All studies reporting using DMEM supplemented it with 10% FBS. In summary, over the past 5 years, the most reported culture media in HL-60 studies was RPMI-10. This is in disagreement with the official protocol from the ATCC, recommending the use of IMDM-20. From 71 studies differentiating HL-60, 65 (>90%) used DMSO as differentiating agent. Over half of DMSO protocols reported supplementing with 1.3% for neutrophilic differentiation purposes (55.4%, Fig. 1d ) followed by 1.25% (30.7%). The length of differentiation varied greatly from 1 to 9 days prior to functional assays ( Fig. 1e ). However, most studies fell within the range of 5-7 days (66.1% of studies reporting DMSO differentiation). Taken together, this systematic review of the recent literature highlights a lack of consensus for HL-60 maintenance culture and differentiation protocols. Therefore, we compared the 3 most reported maintenance media for HL-60: RPMI-10, IMDM-20, and DMEM-10. For neutrophilic differentiation, the consensus 1.3% DMSO differentiation stimulus was applied over 5 or 7 days of culture. Both RPMI-10 and DMEM-10 during neutrophilic differentiation reduced fold expansion and viability. We investigated the impact of culture media and length of differentiation on neutrophil-like cells. Using IMDM-20 significantly increased total fold expansion irrespective of length of differentiation (p <0.0001, Fig. 2a ) while fold-expansion was similar using DMEM-10 or RPMI-10. Consequently, using IMDM-20 lead to significantly more neutrophil-like cells compared to using DMEM-10 and RPMI-10 at day 5 (p =0.0004 and <0.0001, respectively) and at day 7 ( Fig. 2a ). Viability decreased significantly at day 5 compared to day 0 in all 3 media ( Fig. 2b ), consistent with the lower viability of post-mitotic cells. Using IMDM-20 led to significantly higher viability during differentiation, compared with using DMEM-10 or RPMI-10 at day 5 (p =0.0001 and <0.0001, respectively, Fig. 2b ) and day 7 (p <0.0001 for both). There was no difference in viability at either day 5 and 7 between DMEM-10 and RPMI-10. Increased FBS in culture has been linked to higher cell proliferation 11,12 . The content of non-essential amino acids is higher in IMDM compared to RMPI and DMEM ( Table 1 ). For example, alanine is only present in IMDM, and asparagine, aspartic acid, and proline are all present in higher amounts in IMDM. IMDM and DMEM have a higher glucose content compared to RPMI ( Table 1 ). RPMI also contains less glucose compared to DMEM and IMDM. Others have shown that higher glucose in in vivo models correlates with increased myelopoiesis 13 . Overall, we propose that the observed higher yields in IMDM-20 are due to multifactorial and possibly synergistic metabolic effects, resulting from the increased serum, glucose, and non-essential amino acids in this medium. This observation urges more research into metabolic requirements during neutrophil differentiation for rational media design. CD15 + CD11b + co-expression was significantly lower in DMEM-10-differentiated cells. We then asked if media choice could impact the neutrophil-like phenotype of differentiated HL-60 cells. We evaluated the proportion of CD15 + CD11b + cells in HL-60 cells, and at day 5 and 7 of differentiation for each media tested ( Fig. 2c ). Compared to HL-60, we observed a significant increase in CD15 + CD11b + at day 5 (p differentiation (p <0.0001 for IMDM and RPMI, and p =0.0001 for DMEM). For both length of differentiation, DMEM-10 cultured-cells had significantly lower proportions of CD15 + CD11b + cells compared to IMDM-20- and RPMI-10-cultured cells ( Fig. 2c , p RPMI-10 and IMDM-20. Therefore, over 5 days of DMSO-mediated differentiation, the proportion of neutrophil-like cells increases irrespective of the type of culture media. Others showed that neutrophils maturation from promyelocytes in vitro takes 7 days, but HL-60 cells are heterogenous and the proportion of neutrophil-like cells increases after 5-7 days 4,14 . Using DMEM-10 led to the lowest proportion of neutrophil-like cells at both lengths of differentiation. It is possible that the lack of vitamin B12 in DMEM-10 contributed to poorer neutrophilic maturation ( Table 1 ). Indeed, vitamin B12 deficiency correlates with impaired neutrophil differentiation, mimicking acute leukemia 15,16 . Phagocytosis was unaffected by media choice, but DMEM-10 correlated with lower bactericidal activity, and RPMI-10 correlated with higher ROS production ability. Media choice has been linked with phenotypic alterations of monocyte-derived macrophages and monocyte-derived dendritic cells 17,18 . Here, we asked if major neutrophilic antimicrobial functions were affected by the media used during in vitro differentiation. The proportion of phagocytic cells was similar across media types and differentiation length ( Fig. 3a ). Median APC + fluorescence was also similar across conditions, suggesting similar efficiency of phagocytosis ( Fig. 3b ). Using DMEM-10 correlated with a decrease in neutrophil-like phenotype, as suggested by significantly lower CD15 + CD11b + proportions. This may seem inconsistent with similar phagocytic efficiency, but immature/band neutrophils, present in HL-60 cultures, can also perform this function 19 . We measured a significant increase in ROS production capacity in cells cultured in RPMI-10 for 5 days compared to IMDM-20 and DMEM-10 at 5 and 7 days ( Fig. 3c ). Using RPMI-10 during a 7-day differentiation did not produce cells with higher ROS production capacity however, and this may be explained by the decreased viability of the culture at day 7 ( Fig. 2b ). Therefore, differentiating neutrophil-like cells in RPMI-10 over 5 days leads to cells with an enhanced capacity to produce ROS compared to using IMDM-20 or DMEM-10. ROS production is significantly reduced in neutrophils when the culture is supplemented with N-acetyl cysteine and glutathione 20 . Although RPMI contains glutathione ( Table 1 ), we hypothesize that the lower ROS production capacity measured in IMDM-20 and DMEM-10 media could be linked to higher media concentrations of cystine, a glutathione precursor 21 . We finally asked if media choice impacted direct bacterial killing. We co-cultured cells with an E. coli clinical isolate. RPMI-10 and IMDM-20 were able to clear efficiently bacteria after 90 minutes of co-culture, irrespective of the length of differentiation (>99%, Supplementary Fig. 2a ), while cells produced in DMEM-10 did not. As these assays were performed in PBS, there was no direct impact of culture media on bacterial growth. Physiological profile of cytokines production from IMDM-20 differentiated HL-60 cells. Neutrophils produce a range of cytokines upon activation 22 . We investigated if media choice affected cytokines production by neutrophil-like cells. We co-cultured neutrophil-like cells for 8 hours with immune complexes (IC) and measured various proinflammatory cytokines in culture supernatants. Using DMEM-10 led to supernatants containing significantly more IL-8 compared to using IMDM-20 over 5 or 7 days (p =0.0071 and 0.0077, respectively, Fig. 3d ). At day 7, using RPMI-10 led to the lowest IL-8 production compared to IMDM-20 and DMEM-10 (p =0.0109 and 0.0038, respectively, Fig. 3d ). Comparing between days, RPMI-10 and DMEM-10 supernatants contained significantly less IL-8 at day 7 compared to day 5 (p =0.0002 and p =0.0224, respectively, Fig. 3d ), which correlate with cell viability. These results show that IL-8 concentration following stimulation varied depending on the media used. IL-6 production is prompted by cellular activation, for example upon FcγRs crosslinking with the Fc portion of antibodies from IC 23 . Using IMDM-20 led to significantly more IL-6 in supernatants after IC stimulation, compared to using RPMI-10 or DMEM-10 at day 5 and 7 of differentiation ( Fig. 3e ). Additionally, using IMDM-20 led to a significant increase in IL-1β production compared to using RPMI-10 or DMEM-10 ( Fig. 3f ). IL-1β is produced after LPS stimulation 24 . It is possible that LPS from E. coli used to produce the IC could protrude and activate neutrophil-like cells. The increased production seen in IMDM-20 compared to RPMI-10 could be due to its higher glucose content. Neutrophils from diabetic patients showed an increase in IL-1β production when stimulated 25 . Although DMEM-10 and IMDM-20 have the same glucose concentration, we propose that the lower IL-1β concentration may be explained by the significantly lower proportion of CD15 + CD11b + cells produced in this media. There was no difference in TNF-α and IL-10 concentration across media ( Supplementary Fig. 2b-c ). TNF-α production depends on phospholipase A2 26 and complement signaling 27 . However, these may not have been involved in the present in vitro assay, as the pooled sera used for opsonization were heat-inactivated before use. Taken together, these results suggest that using IMDM-20 leads to neutrophil-like cells that could secrete IL-1β and IL-6 upon IC-mediated activation, while using RPMI-10 and DMEM-10 produces cells with a lower cytokine production capacity. These results invite to study how cytokines production can be regulated by culture metabolites composition. Conclusions Culture media type has been linked to alterations in immune cell lines phenotypes and functions 17,18,28,29 . Here, we show that using IMDM-20 over 5-7 days of differentiation with 1.3% DMSO yields high cell numbers, and neutrophil-like cells with phagocytosis and ROS production capacity, a varied cytokines response, and an efficient pathogen clearance. The present work describes the use of IMDM-20 and 1.3% DMSO to produce a better surrogate model compared to other media options. Systematic search strategy A systematic search of the literature available in the PubMed platform published between January 9 th , 2020, and January 9 th , 2025 included, on HL-60 derived neutrophil-like cells was performed. We conducted the search using the keywords “neutrophil-like cell*” and “HL-60” ( Supplementary table 3 ). We included original research papers written in English that described HL-60 cells culture. Charted metadata included culture media, type and proportion of serum supplementation, reagent used for differentiation and its concentration, and length of culture in differentiation media. HL-60 culture HL-60 (American Type Culture Collection, Manassas, VA, USA, CCL-240) passage 9 were revived in IMDM (Gibco,Waltham, MA, USA, cat. 12440046), supplemented with 20% FBS (Gibco, cat. 26140079), as per ATCC recommendations. Cells were manually counted using a Neubauer chamber with trypan blue (Gibco, cat. 15250061) exclusion for viability assessment. Cells were adapted to RPMI (Merk, Darmstadt, Germany, cat. R4130) supplemented with 10% FBS, or DMEM (Sigma-Aldrich, St. Louis, MO, USA, cat. D5796) supplemented with 10% FBS. All media were supplemented with 1% of Antibiotic/Antimycotic (Gibco, cat. 15240062). Media adaptation was performed at passaging every 3 days, as follow: with cell viability >90%, fresh culture media was added including 40% new media composition compared to the last passage. If cell viability <90%, cells were passaged using the same media composition used at the previous passage. Differentiation towards neutrophil-like cells was performed at a cell density of 4 x10 5 cells/mL using 1.3% of DMSO (Sigma-Aldrich, cat. D2650) during 5 or 7 days. All experiments were performed before passage 35. No testing of mycoplasma was performed. Flow cytometry To investigate relative surface markers expression, the following monoclonal antibodies were used: anti-human CD11b-AF700 (BD Biosciences, San Jose, CA, USA, cat. 557918, clone ICRF44) and CD15-BV786 (BD Biosciences, cat. 563838, clone HI98), at titrated concentrations. For viability assessment, propidium iodide was added 15 min before acquisition, as per manufacturer’s instructions. Samples were acquired immediately after staining on a FACSCelesta flow cytometer fitted with 405 nm, 488 nm, and 640 nm lasers and operated through the BD FACSDiva software v.8. Cytometer settings were validated as within the range of manufacturer’s recommendations using CS&T beads (BD Biosciences, cat. 642412) prior to each acquisition. Compensation controls were used at each acquisition using the CompBeads anti-mouse Ig, κ/Negative control compensation particle set (BD Biosciences, cat. 552843) following manufacturer’s recommendations. At least 10,000 live singlets were acquired per sample. Flow cytometry .fcs3 files were analyzed using FlowJo v.10 (BD Biosciences). The gating strategy ( Supplementary Fig. 3 ) was based on FMO controls used to adjust gates. Flow cytometry data are publicly available in the Zenodo repository (Zenodo, CERN, Geneva, Switzerland) at: https://doi.org/10.5281/zenodo.15258439. Phagocytosis We used pHrodo™ Deep Red E. coli BioParticles™ (Invitrogen, Waltham, MA, USA, cat. P35360) to produce immune complexes (IC) for phagocytosis. Briefly, 20 µL of donor pooled sera (n =7 donors) were used to opsonize 20 µL of bioparticles for 30 min at 37 °C, with 100 rpm agitation. Then, 1.75 x10 5 HL-60 derived neutrophil-like cells were challenged with the IC for 30 mins at 37 °C and 5% CO 2 without agitation at a 1:3 V/V. After incubation, cells were analyzed immediately using flow cytometry, as described above. The gating strategy is presented in Supplementary Fig. 4 . ROS production ROS production was evaluated by the oxidation of 1,2,3-dihydrorhodamine (DHR) (Invitrogen, cat. D23806). Briefly, 1 µM DHR in 2 x10 5 cells in 1 mL of PBS were stimulated with 100 nM PMA for 15 min at 37 °C. After incubation, the reaction was stopped with a 10 min incubation on ice and cells were washed with PBS at 300 rcf for 5 min at 4 °C. DHR fluorescence was measured immediately by flow cytometry by measuring MFI at 530 nm. The stimulation index was determined as the ratio of PMA-stimulated-cells MFI to unstimulated cells to normalize for background DHR cleavage. The gating strategy is presented in Supplementary Fig. 5 . Cytokines production Cytokines released in culture supernatant after stimulation with IC were measured by flow cytometry using a Human Inflammatory Cytokine CBA Kit (BD Biosciences, cat. 551811). Briefly, IC were produced by incubating 1.3 x10 8 UFC/mL UV-killed E. coli DH5a cells with 40 μL of 50% donor pooled sera (n= 10 donors) during 30 min at 37 °C at 100 rpm agitation. Then, 3 x10 5 HL-60 derived neutrophil-like cells were co-incubated with IC for 8 hours at 37 °C and 5% CO 2 without agitation. Culture supernatants were collected and frozen at -80 °C until analysis. Experiments were run on 3 separate occasions. Bacteria killing assay E. coli 066 30 cells were washed with PBS at 10,000 rcf for 10 min at 4 °C. Bacteria were then resuspended in 1 mL of opsonizing media (PBS + 10% pooled sera from 10 donors) for 30 min at 37 °C at 24 rpm in a Mini Labroller TM rotator (Labnet International, Edison, NJ, USA). One million neutrophil-like cells were co-cultured with opsonized bacteria at a bacteria to cell ratio of 5:1 in 500 µL of PBS. Bacteria without added neutrophils were used in each experiment as controls. Co-cultures were performed at 37 °C and 24 rpm for 90 minutes. Culture dilutions were then plated in LB agar plates overnight and CFU were counted the following day. Killing was assessed as reduction of bacterial after co-incubation, calculated by subtracting the log 10 of bacteria only from the log 10 of bacteria co-incubated with neutrophil-like cells. Statistical analysis Data distributions were determined using Shapiro-Wilk tests. When data were normallydistributed, two-way ANOVA tests were used to evaluate the impact of media and differentiation times. Post-hoc tests were applied to identify significant differences, as relevant described in figure legends. All statistical tests were performed using Prism v.10 software (GraphPad Software, San Diego, CA, USA). p values <0.05 were considered statistically significant. Error bars indicate standard deviation and n values represent biological replicates. AUTHORSHIP JACP: Conceptualization, Methodology, Formal analysis, Investigation, Writing-Original draft, Writing–Review & Editing. 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A systematic search of the literature published between January 9, 2020, and January 9, 2025 was performed in PubMed. Original research articles reporting HL-60 cell culture protocols were included (n = 75). (a) Type of culture media reported in included studies. (b) FBS% in IMDM maintenance culture media reported in included studies. (c) FBS% in RPMI maintenance culture media reported in included studies. (d) Number of studies organized according to the distinct DMSO concentrations used to induce neutrophil-like differentiation. (e) Reported days of culture in DMSO-differentiation media to induce neutrophil-like differentiation. FBS: fetal bovine serum, FCS: fetal calf serum, DMSO: dimethyl sulfoxide. Figure 2. Differentiating HL-60 cells in IMDM-20 led to more neutrophil-like cells with increased viability. (a) Absolute fold expansion during HL-60 differentiation with 1.3% DMSO on day 5 and 7 post-induction (n = 5). (b) Viability of differentiated HL-60 cells at day 5 and 7 post-induction (n = 5). (c) Proportion of cells co-expressing CD15 + CD11b + in HL-60 and at day 5 and 7 after 1.3% DMSO (n = 3). Data presented as a pool of biological replicates analyzed on separate days. Data is presented as mean and standard deviation. Two-way ANOVA tests were used with Tukey post-hoc to compare media and length of differentiation. ****p<0.0001. DMSO: dimethyl sulfoxide. Figure 3. Phagocytosis, ROS production, cytokines production and killing efficiency according to media type and length of neutrophilic differentiation. (a) Proportion of APC + phagocytic cells after APC- Escherichia coli bioparticles phagocytosis (n = 2-4). (b) MFI of APC + population (n = 2-4). (c) ROS production index, calculated by dividing FITC MFI from PMA stimulated cells by the MFI from non-stimulated cells (n = 3-5). (d) IL-8 concentration in supernatant after IC stimulation for 8 hours (n = 3). (e) IL-6 concentration in supernatant after IC stimulation for 8 hours (n = 3). (f) IL-1β concentration in supernatant after IC stimulation for 8 hours (n = 3). Data presented as a pool of biological replicates analyzed on separate days. Data is presented as mean and standard deviation. Two-way ANOVA tests were used with Tukey post-hoc to compare between media type and length of differentiation in the distinct neutrophilic functions assessed. *p<0.05, **p<0.01, ***p<0.001. MFI: median fluorescence intensity, DHR: dihydrorhodamine 123, PMA: phorbol 12-myristate 13-acetate. Supplementary Material File (figures 1-3.pdf) Download 217.06 KB Information & Authors Information Version history V1 Version 1 23 April 2025 Peer review timeline Published Biochemistry and Biophysics Reports Version of Record 1 Sep 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords culture media differentiation hl-60 neutrophils Authors Affiliations Jorge Andrés Cázares-Preciado 0000-0003-2661-584X Tecnologico de Monterrey Escuela de Ingenieria y Ciencias View all articles by this author José Antonio Cruz-Cardenas Tecnologico de Monterrey Escuela de Ingenieria y Ciencias View all articles by this author Alejandra López-Arredondo Tecnologico de Monterrey Escuela de Ingenieria y Ciencias View all articles by this author Marco Vinicio Gallardo-Camarena Tecnologico de Monterrey Escuela de Ingenieria y Ciencias View all articles by this author Marion E. G. Brunck [email protected] Tecnologico de Monterrey View all articles by this author Metrics & Citations Metrics Article Usage 376 views 167 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jorge Andrés Cázares-Preciado, José Antonio Cruz-Cardenas, Alejandra López-Arredondo, et al. IMDM-20 enhances neutrophilic features during DMSO-mediated differentiation of HL-60 cells. 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