Alexa fluor 647 conjugated anti mouse cd11b clone m1 70

The stimuli used for treating cells were: GM-CSF (Shenandoah, 200-15), CpG ODN 1826 (InvivoGen, tlrl-1826), CL097 (InvivoGen, tlrl-c97), fMLF (Sigma), PMA (Sigma). The antibodies used for flow cytometry staining are as follows: FITC-conjugated anti-Ly6G (clone 1A8, BD biosciences) and Alexa Fluor 647-conjugated anti-mouse CD11b (clone M1/70, BD biosciences). The anti-Nlrp3 antibody used in this study was from R&D (MAB7578), and detects both human and mouse proteins. Nexinhib20, 4,4-dimethyl-1-(3-nitrophenyl)-2-(1H-1,2,4-triazol-1-yl)pent-1-en-3-one was described previously, its molecular signature and purity were confirmed by mass spectrometry analysis (Johnson et al., 2016 (link)). Nexinhib20 was dissolved at a concentration of 10 mM in DMSO and diluted in PBS. The final concentration of Nexinhib20 in the reaction media was 10 μM.

To stain for cell-surface markers, cells were blocked in ice-cold PBS containing 1% BSA, and stained with fluorescein isothiocyanate (FITC)-conjugated anti-Ly6G (clone 1A8, BD biosciences) and Alexa Fluor 647-conjugated anti-mouse CD11b (clone M1/70, BD biosciences). The cells were then washed and fixed in 1.5% paraformaldehyde in PBS. The samples were analyzed using a BD LSR II flow cytometer (BD Biosciences) and the data was analyzed using FlowJo software.