Fluorescence Microscopy of BMDM Infection

For fluorescence microscopy experiments, BMDMs (0.5 × 10⁶) were seeded in a 4-chamber μ-slide (80426, Ibidi). Chambers were infected as described above. Wells were aspirated and fixed in 4% paraformaldehyde (PFA), washed with PBS, permeabilized with 0.1% Triton X-100, and stained with phalloidin-iFluor647 (ab176759, Abcam), and DAPI (4′,6-diamidino-2-phenylindole). For confocal images, a Nikon C2 microscope was used as previously described (59 (link)). Quantitative cell death measurements by SYTOX Green or PI uptake counts were collected hourly on an IncuCyte S3 system (Essen BioScience). Within each replicate well (on a 96-well plate), nine images were collected, and cell death was quantified using the Basic Analyzer software of the IncuCyte S3 and exported as “object count per well,” which extrapolates the “object count” from the average count across images to the total well area. At least four replicate wells were included for each experiment, and these replicate wells were used to determine statistical significance between treatment conditions.