Peripheral blood mononuclear cells isolation

To assess differences in the level of expression and activation of the NLRP3 inflammasome between GC‐resistant versus GC‐sensitive patients, we collected 15 ml of peripheral blood from 3 GC‐resistant and 3 GC‐sensitive adult patients with FSGS with INS at the time of diagnosis. Peripheral blood mononuclear cells (PBMCs) were isolated by density separation over a Ficoll–Paque (GE Healthcare, Chicago, IL) gradient (460 g for 30 min) and washed 3 times with phosphate‐buffered saline (PBS) pH 7.4/1 mM EDTA (Sigma‐Aldrich, St. Louis, MO). Due to the difficulty in obtaining biological samples of GC‐resistant patients, we considered to compare at least three resistant versus thee sensitive patients.