Differentiation of HL-60 cells in serum-free hematopoietic cell media enhances the production of neutrophil extracellular traps

Neutrophil extracellular traps (NETs) are web-like structures made of chromatin and have been identified to have a role in the host's immune defense. Differentiated human promyelocytic leukemia HL-60 cells (dHL-60) have been used to study the mechanisms of NETs formation, as neutrophils have a short lifespan that limits their use. However, dHL-60 cells are inefficient at generating NETs and therefore are not ideal replacements for neutrophils in studying of NET formation. In the present study, the optimal cell culture conditions and differentiation time that result in the most effective release of NETs from dHL-60 cells upon stimulation were determined. HL-60 cells were cultured in serum (FBS) or serum-free (X-VIVO) medium and differentiated using all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). dHL-60 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) or Ca(2+) ionophore (CI). Cell differentiation and apoptosis, as well as the formation of reactive oxygen species (ROS) and citrullinated histone H3 (citH3) were analyzed using flow cytometry. NETs were visualized using fluorescence microscopy and NET quantification was performed using PicoGreen. Induction of HL-60 cells for five days produced the best results in terms of differentiation markers and cell viability. Both ATRA- and DMSO-induced dHL-60 cells were able to release NETs upon PMA and CI stimulation; dHL-60 cells in serum-free medium produced more NETs than those in serum-containing medium. DMSO-dHL-60 (X-VIVO) cells were most efficient at producing NETs and ROS upon stimulation with PMA, while ATRA-dHL-60 (X-VIVO) cells were most efficient at producing NETs and citH3 upon stimulation with CI. It was concluded that DMSO-dHL-60 (X-VIVO) may be a model for the study of ROS-high NETosis and ATRA-dHL-60 (X-VIVO) may be suitable for ROS-low NETosis.