Bio plex pro human cytokine assay

iPS-MLs or iPS-MPs were harvested as described above and seeded into 96-well plates at 5 × 10⁴ cells per well. When indicated, cells were pretreated with ABT-751 (10 µM; Sigma-Aldrich), CA4P (10 µM; Sigma-Aldrich), MCC950 (10 µM; Sigma-Aldrich), and colchicine (100 ng/ml; Sigma-Aldrich) 30 min before LPS priming or with 2BP (20 µM; Sigma-Aldrich) and bryostatin (0.1 µM; Sigma-Aldrich) 24 h before harvest. For inflammasome activation, cells were primed with LPS (1 µg/ml) for 4 h and treated with UCN-01 (10 µM; Sigma-Aldrich), nigericin (10 µM; Sigma-Aldrich), or purified flagellin from Salmonella typhimurium (InvivoGen) in DOTAP liposomes (Roche; IC-FLA; 6 µg DOTAP/1 µg FLA-ST) for an additional 2 h, after which the supernatants were collected. For TcdA stimulation, TcdA (1 µg/ml; List Biological Laboratories) was added after 2 h of LPS priming (1 µg/ml) and the supernatants were collected 4 h later. For AIM2 inflammasome activation, poly (dA; dT)/Lyovec (1 µg/ml; InvivoGen) was added along with LPS priming (1 µg/ml), and the supernatants were collected 4 h later. The IL-1β, IL-18, and IL-6 concentrations were measured using the Bio-Plex Pro Human Cytokine Assay (BioRad).