Live dead fixable aqua dead cells stain

PBMCs were incubated with LIVE/DEAD Fixable AQUA Dead Cells Stain (Thermo Fisher, USA) for 15 min at RT, followed by extracellular staining in Gelatin Veronal Buffer (GVB) (CompTech complement Technology Inc, Tyler, TX) for 30 min at room temperature (RT) to characterize complement staining in monocytes with the following monoclonal antibodies (clones) and fluorochromes: CD14 (M5E2) BV605, CD16 (3G8) PE-Cy7, CD56 (HCD56) BV421, CD66b (G10F5) Pacific Blue from Biolegend, HLA-DR (L243) APC-Cy7, C3 (1H8) PE and CD59 (p282(H19)) BV711 from BD bioscience, CD2 (RPA-2.10) eFluor 450, CD3 (UCHT1) eFluor 450, CD19 (SJ25C1) eFluor 450 and CD20 (2H7) eFluor 450 from Life Technology and, finally, C1q (polyclonal) FITC from Dako. Data were acquired on a BD Fortessa flow cytometer (BD Biosciences). All compensation and gating analyses were performed using FlowJo 10.5.3 (TreeStar, Ashland, OR, USA).

Inflammasome complex assembly was evaluated by detection of ASC speck formation by imaging flow cytometry, as previously described [32 (link)]. Briefly, cells were incubated with the fluorochrome inhibitor of caspase-1/4/5 (FLICA, from Immunochemistry technologies (ICT), Bloomington, MN) following the manufacturer’s instructions, for 50 min at 37°C, to allow for binding of FLICA to activated inflammatory caspases. Cells were washed twice with FLICA wash buffer and then incubated with LIVE/DEAD Fixable AQUA Dead Cells Stain (Thermo Fisher, Massachusetts) for 15 min at RT, followed by extracellular staining in PBS + 1% BSA with fluorochrome-conjugated antibodies for monocyte phenotyping as listed in S3 Table. Cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 1h at RT and stained with anti-ASC/TMS1 antibody from Novus Biologicals, CO, USA, for another 1 hr. Samples were washed twice and resuspended in 50 μL of PBS+1%BSA.