An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

For immunofluorochemical analysis of the nigericin-treated cells, wash the probe-labeled cells, and incubate the cells in 250 microliters of fixation and permeabilization solution per well for 30 minutes on ice, protected from light.

Wash the macrophages three more times with fresh wash buffer, and label the cells with 250 microliters of primary antibody against apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain, or ASC, per well for one hour on ice.

Wash the cells at the end of the incubation, and label the samples with 250 microliters of the appropriate fluorescent dye-conjugated secondary antibody for another hour on ice, adding 4 microliters of an appropriate fluorescent nuclear staining dye during the last 5 minutes.

After washing the cells 3 times in cold wash buffer, mount coverslips onto the slides with 7 microliters of anti-fade mounting medium as demonstrated, and image the macrophages by fluorescence confocal microscopy.