Investigating Neutrophil Function with NB4 Cells

NB4^PMN cells were used to study the effect of RETN on neutrophil function. The NB4^PMN cell line offers the advantage of standardization and reproducibility when compared with primary neutrophils [9 (link), 15 (link)]. Neutrophilic differentiation was achieved as previously described [9 (link)]. Briefly, NB4 cells were treated with 1 μM all-trans retinoic acid (ATRA) for 6 days in Roswell Park Memorial Institute medium (RPMI 1640) (VWR, Radnor, PA) with l-glutamine containing 10% fetal bovine serum (FBS) (Atlanta Biologicals; Flowery Branch, GA). Successful neutrophil differentiation was assessed by flow cytometry to monitor expression of CD11b, CD35, and CD71 surface markers (BD Biosciences, San Jose, CA). In some experiments, NB4^PMN cells were incubated for 1 h in human control serum (Atlanta Biologicals) containing 100 ng/mL of recombinant human resistin (R&D Systems, Minneapolis, MN) to replicate concentrations measured in patients with sepsis-induced AKI [15 (link)].