Immunostaining of Macrophages After Bacterial Stimulation

Macrophages were plated on glass coverslips. After bacterial stimulation, coverslips were washed with PBS, fixed in 2% PFA, and quenched in 50mM NH₄Cl. Cells were then permeabilized with 0.2% Triton X-100 in PBS before blocking with 10% FBS PBS and incubation with appropriate antibodies prepared in 10% FBS PBS. When indicated, Phalloidin Alexa 647 was used together with secondary fluorescent antibodies to stain Actin and delineate the cell area. Lastly, cells were stained with DAPI. Coverslips mounted with ProLong Diamond Antifade Mountant (Invitrogen). Images acquired on a Leica SP5 DM microscope with a 63x/1.4 NA oil immersion objective. The partial nuclear staining pattern noted in Extended Data Figure 4 has been reported^{60 (link)} and is ASC-specific given its absence in Pycard^–/– macrophages.