PBMCs were treated with either nothing or E. coli LPS serotype 055:B5 (Sigma-Aldrich, USA; 1 μg/mL, 2 h at 37 °C). LPS-primed PBMCs were then stimulated with either nothing, ATP (3 mM), or nigericin (10 μM) for 30 min at 37 °C. Stimulated PBMCs were fixed with 2% paraformaldehyde (Sigma-Aldrich, USA) and stained for the detection of intracellular ASC specks by TOFIE as previously described [12 (link)], using the rabbit polyclonal antibody anti-ASC (N-15)-R (Santa Cruz Biotechnology, USA).
For active caspase-1 detection, PBMCs were incubated for 1 h with FLICA660 reagent (ImmunoChemistry Technologies, USA) and fixed following manufacturer recommendations. Monocytes were detected with the APC-vio770-conjugated anti-human CD33 antibody (Miltenyi Biotec, Germany) and with the APC-Cy7-conjugated anti-human CD14 antibody (TONBO Biosciences, USA). Stained cells were acquired on a FACSCanto cytometer (BD Biosciences, USA). Cytokines were measured in cell supernatants using a custom bead-based multiplex Luminex immunoassay (eBioscience, USA). Heat maps representing cytokine expression profiles were created using Morpheus software (Broad Institute, Cambridge, USA).
For active caspase-1 detection, PBMCs were incubated for 1 h with FLICA660 reagent (ImmunoChemistry Technologies, USA) and fixed following manufacturer recommendations. Monocytes were detected with the APC-vio770-conjugated anti-human CD33 antibody (Miltenyi Biotec, Germany) and with the APC-Cy7-conjugated anti-human CD14 antibody (TONBO Biosciences, USA). Stained cells were acquired on a FACSCanto cytometer (BD Biosciences, USA). Cytokines were measured in cell supernatants using a custom bead-based multiplex Luminex immunoassay (eBioscience, USA). Heat maps representing cytokine expression profiles were created using Morpheus software (Broad Institute, Cambridge, USA).