Phagocytosis of PS-exposed-necroptotic cells.

<p><b>(A)</b> U937 cells were first stained with CFSE prior to stimulation for apoptosis and necroptosis using a combination of TNFα, birinapant (SMAC mimetic), and zVAD. PS exposure was tested every 30 minutes until exposure reached 40% in both the apoptotic and necroptotic samples (determined by A5/PI staining). Cells were washed twice and resuspended in DMEM before adding on IFN-γ treated BMDMs at a 2.5:1 ratio. Phagocytosis was analyzed by flow cytometry (mean ± SD). BMDMs with or without addition of live cells served as negative controls. Data are taken from 3 independent experiments. <b>(B)</b> U937 cells treated as above were added on TG peritoneal macrophages at a 2.5:1 ratio, and phagocytosis was analyzed by flow cytometry (mean ± SD). <b>(C)</b> Viability of the U937 cells, which were used as target to (B), is shown. TG peritoneal macrophages with no addition of cells or with addition of live cells served as negative controls. <b>(D–I)</b> Supernatants from the phagocytic TG peritoneal macrophages from (B) were collected and then analyzed for cytokines and chemokines using ELISAs. Statistic comparisons between each injected target cells were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. <b>(J–K)</b> U937 cells were left untreated or stimulated for apoptosis or necroptosis. PS exposure was tested every 30 minutes until reaching 40% in apoptosis and necroptotic samples (determined by A5/PI staining). 2 x 10⁶ cells in 100 μl per mouse were IP injected. One hour after injection, CD11b peritoneal cells were analyzed for CFSE fluorescence <b>(J)</b> and phagocytosis of CFSE target cells <b>(K)</b> by flow cytometry (<i>N</i> = 4, mean ± sem). Statistic comparisons between each injected target cells were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. <b>(L)</b> Illustration of competitive in vivo phagocytosis assay. CFSE- or Hoescht-stained L929 cells were left untreated or stimulated for necroptosis (TSZ). PS exposure was tested every 30 minutes until reaching 40% in necroptotic samples (determined by A5/PI staining). A 1:1 ratio mix of live and necroptotic cells was IP injected (total of 2 x 10⁶ cells in 100 μl per mouse). (M) One hour after injection, F4/80 peritoneal cells were analyzed for phagocytosis of the live and/or necroptotic cells by flow cytometry (<i>N</i> = 4, mean ± sem). Statistic comparisons between each injected target cells populations were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s013" target="_blank">S6 Data</a>. A5, annexin V; BMDM, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; DMEM, dulbecco’s modified eagle’s medium; IP, intraperitoneally; PI, propidium iodide; MFI, mean fluorescence intensity; PS, phosphatidylserine; SD, standard deviation; SMAC, second mitochondrial-derived activator of caspase; TG, thioglycolate; zVAD, Z-VAD-FMK.