Gating strategies and validation of protocol. (A) PBMCs isolated from healthy donors were treated for 1 h with FAM–YVAD–FLICA before CD14 labeling and then analyzed by flow cytometry. Live cells were gated and doublets were excluded using SSC-A and SSC-H plot. CD14 high monocytes were analyzed for the presence of Caspase-1 speck using FITC-Caspase-1-A and FITC-Caspase-1-H. Monocytes with Caspase-1 specks were defined by a high Caspase-1-A/Caspase-1-H ratio. (B) PBMCs were treated 8 h with LPS (100 ng/ml) and 1 h with Nigericin (5 µM) (LPS + Nigericin) or with vehicle (Control). After 1 h incubation with the FAM–YVAD–FLICA probe cells were CD14 labeled and analyzed by flow cytometry. Experiments were repeated at least four times and representative data are shown. (A and B) The data were reused from Fig. 6 A. (C) Graphic representation showing the percentage of cells with Caspase-1 speck. (D) Overlay of the FAM–YVAD–FLICA fluorescence intensity measured in monocytes healthy donors (Control) and cells treated with LPS and Nigericin (LPS + Nigericin). (E) Graphic representation of data showing the MFI measured. Data are expressed as the mean ± SEM. Statistical analyses were performed using paired t test. *P < 0.05. Data are representative for n = 3.