An optimized method to differentiate HL60 cells into neutrophil-like cells

The HL60 promyelocytic leukemia cell line is widely used to investigate neutrophil biology due to its genetic tractability and accessibility. HL60 cells can be differentiated into neutrophil-like cells using all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO) treatment. However, these approaches produce cells that lack critical features of mature granulocytes, such as robust chemotaxis with ATRA treatment or multilobed nuclear morphology with DMSO treatment. To overcome these limitations, we developed a sequential differentiation protocol-ATRA for 1 d followed by DMSO for 4 d (A1D4)-which yields neutrophil-like cells that more faithfully recapitulate the morphology and functionality of mature human neutrophils. A1D4-differentiated HL60 cells display segmented nuclei with low lamin A/C expression and demonstrate strong chemotactic, oxidative burst, and phagocytic activity. This protocol combines the advantages of treatment with the individual compounds, producing cells that closely mimic primary human neutrophils in both phenotype and function.