Caspase-1 activation and active caspase-1 foci formation was determined by FAM-YVAD-FMK (ImmunoChemistry Technologies) staining of macrophages infected on glass coverslips. DNA was stained with Hoescht 33342. Cells were washed with PBS and subsequently fixed with Cytofix (BD Biosciences). Immunofluorescence staining was performed after fixation in Perm/Wash (BD Biosciences) with antibodies specific to NLRP3 (sc-66846, Santa Cruz Biotechnology), ASC (ADI-905-173-100, Enzo Life Sciences), or pro–caspase-1 (sc-514, Santa Cruz Biotechnology) with Alexa Fluor–conjugated secondary antibodies (Invitrogen). Coverslips were mounted with ProLong (Molecular Probes) and examined using confocal microscope (Leica SP8X) at the W. M. Keck Microscopy Center. Caspase-1 activation or foci formation was enumerated by counting the fraction of positive cells in four separate fields for each coverslip. Caspase-1 and ASC foci colocalization was enumerated by counting the fraction of positive cells in eight separate fields from duplicate coverslips.

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