Differentiation of HL-60 Cells into Neutrophil-like Cells

The human promyelocytic leukemia cell line HL-60 (ATCC CCL-240) was obtained from the American Type Culture Collection (ATCC). HL-60 cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM; Gibco) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 1 mg/mL streptomycin and cultured in an incubator at 37°C with 5% CO₂. Cells were routinely counted to maintain a density of between 10⁵ and 10⁶ cells/mL. HL-60 cells at a density of 10⁶ cells/mL were differentiated into neutrophil-like cells using growth medium supplemented with 1.25% dimethyl sulfoxide (DMSO) and incubated for a period of 5 to 7 days. Prior to use in the assay, the cells were washed in IMDM, and viable cells were counted based on trypan blue exclusion using a hemocytometer. HL-60 cells were then suspended in IMDM plus 1% BSA at a concentration of 2 × 10⁵ cells/mL for use in the opsonophagocytic killing assay (OPA).