Fig. 4

ROS pathway is driving force of cell death and directly involved in FMF. a Neutrophils (5 × 10⁶/ml) from HC (healthy control) and FMF patients were stained with SytoxGreen and incubated for up to 180 min with or without ROS inhibitor DPI (10 μM). Cell death assay as indicated by OD of SytoxGreen was measured for the indicated times (n = 3). b Neutrophils of HC and CGD (chronic granulomatous disease) patients (5 × 10⁶/ml) were stained with SytoxGreen and either left untreated or were stimulated with PMA for up to 4 h. OD of SytoxGreen was measured at the indicated times. Mean and SD of 4 independent experiments is shown. c Supernatants of b (after 4-h culture) were measured for content of S100A12 by ELISA (n = 4). Neutrophils (5 × 10⁶/ml) were incubated up to 120 min harvested and directly stained with dihydrorhodamine (DHR) to visualize ROS directly in flow cytometry (d, n = 6). Supernatants of cell cultures from a (120 min) were measured for S100A12 protein content using an ELISA. Data are mean and SD of 3–6 independent experiments (e). All FMF biosamples were from homozygous patients. Statistical significance was calculated using Brown-Forsythe and Welch’s ANOVA test followed by Dunn’s multi-comparison test (* = p > 0.05, ** = 0.005, *** = p > 0.0005)