IMDM-20 enhances neutrophilic features during 1.3 % DMSO-mediated differentiation of HL-60 cells

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 <i>in vitro</i>. 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 conducted a systematic search of the PubMed database, to investigate the current heterogeneity in published protocols used to differentiate HL-60 towards neutrophil-like cells. Research articles published in English between January 9th^, 2020, and January 9th^, 2025, were identified using the key words "neutrophil-like cell" and "HL-60". Metadata of included research papers were charted and analyzed to evaluate the most reported HL-60 cells culture protocols. 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 opsonized <i>E. coli</i>. Overall, the results support the use of IMDM-20 with 1.3 % DMSO to differentiate HL-60 to study neutrophil biology <i>in vitro</i>.