Ag 25b 006 c100

THP-1 cells were counted and normalized prior to treatment with LPS for 4 h. Cells were washed in PBS, prior to fixation in 4% paraformaldehyde. The samples were loaded into an automated cytospin machine (Thermo Scientific Cytospin 4) following the manufacturer’s instructions and centrifuged (500 rpm for 5 m). Slides were fixed using 4% paraformaldehyde for 10 m, permeabilized with 0.1% Triton-X100 for 10 m, and then blocked with 1% BSA in PBS for 30 m. Cells were probed with primary antibodies ASC (rabbit, AdipoGen, AG-25B-006-C100, 1:50 in 1% BSA in PBS) for 2 h. Following washing, cells were incubated with Alexa Fluor conjugate secondary antibodies (Thermo Fisher, 1:100 dilution for 1 h) and briefly stained with DAPI. Cells were imaged using an Invitrogen EVOS M7000 Imaging System at 40x original magnification. For each biological replicate, three investigators independently counted ASC specks and nuclei in each high power field for at least 200 cells in each condition.