Culturing and Differentiating THP-1 Macrophages

THP-1 cells (ATCC® TIB-202) were maintained as suspension cultures in RPMI 1640 media, supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 1 mg/ml streptomycin, 0.25 μg/ml amphotericin B, 1% ml of 100× non-essential amino acids (Gibco Thermo Fisher), 10 mM HEPES buffer, 1 mM sodium pyruvate, and 2 mM glutamine. Differentiation of THP-1 cells into monolayers of macrophage-like cells was achieved by seeding the cells on plates and stimulating for 24 h with 100 nM phorbol-12-myristate-13-acetate (PMA; EMD Chemicals). THP-1 cells stably expressing an shRNA directed against ASC (47 (link)) or stably expressing a YFP-ASC fusion protein were maintained in RPMI 1640 media, supplemented as above. HEK293 cells (ATCC) and HEK293 stably expressing TLR4-MyD 88 signaling along with YFP-ASC (69 (link)) (Gavrilin et al., personal communication) were maintained in DMEM containing 10% FBS, 100 IU/ml penicillin, 1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B.