Efferocytosis of PIT and associated bacteria in vitro. (A) Dextran–Alexa Fluor 555–labeled WT BMMs were infected with SPI1-induced mCherry-expressing S. Typhimurium as in Video 4 and imaged by live cell microscopy. Images are stills from Video 4 at indicated time points after infection. White arrow indicated a live BMM that efferocytoses a pyroptotic BMM denoted by the arrowhead. (B) WT BMMs were treated with PBS or 3 µg/ml FlaTox for 2 h, labeled with Green CellTracker and quantitated in ImageJ by measuring the total intensity of ∼50 individual live and dead BMMs. (C and D) WT BMMs (Green CellTracker) and Casp1^−/−Casp11^−/− BMMs (Blue CellTracker) were co-cultured and treated with 3 µg/ml FlaTox in PI-containing media while imaged by live cell confocal microscopy. The in vitro percentage of phagocytosis was determined by quantifying the percentage of Casp1^−/−Casp11^−/− BMMs (Blue) that contain CellTracker Green–BMM debris or PI-positive nuclei. (E) CellTracker Orange–labeled and FlaTox-treated WT BMMs were injected i.p into Casp1^−/−Casp11^−/− mice (n = 5). The percentage of live peritoneal macrophages (CD45⁺ CD11b⁺ Ly6G⁻ F4/80⁺) and neutrophils (CD45⁺ CD11b⁺ Ly6G⁺ F4/80⁻) with CellTracker Orange-positive debris was determined by staining and analyzing the peritoneal wash by flow cytometry. All data are representative of three individual experiments. Error bars represent SE. P-values were determined by a two-tailed Student’s t test; *, P < 0.05.