Ells (2 107 cells/ml) were CYP1 Activator manufacturer stained with 250 nM carboxyfluorescein succinimidyl ester (CFSE: Molecular Probes; Life Technologies, Carlsbad, CA, USA) for 1 min. Staining was stopped by the addition of fetal calf serum, and the cells were washed 3 instances with medium. Splenic CD11b+ macrophages from uninfected mice have been sorted with all the MACS cell separation technique, along with the labeled with PKH26, according to the manufacturer’s instructions. Splenic CD11b+ macrophages (1 105 cells) were cocultured with CFSE-labeled pRBCs or normal RBCs inside a 1:30 ratio, at a final volume of 200 l for four hr at 37 within a CO2 incubator with culture medium. Following coculture, the noningested RBCs had been removed with ACK lysing buffer. The capacity of macrophages to phagocytize CFSE-labeled pRBCs or normal RBCs was analyzed with a FACSCalibur flow cytometer. For the in vitro phagocytosis inhibition assay, anti-Tim-4 antibody and its isotype manage antibody were added to the test sample.In vitro PS externalization testSorted erythroid cells (3 105 cells/well) from gld mice 17 days soon after infection with PyNL FP have been cocultured with CD8+ T cells from WT or gld mice 17 days following PyNL infection or from uninfected WT mice at 37 for four hr within a CO2 incubator with culture medium. Effector (CD8): The target (erythroid) ratio was 0:10:1. The cells have been Fc-blocked and stained with PE-Cy7-conjugated anti-TER119 antibody. PS was then stained with PE-conjugated annexin V in calcium-containing annexin V binding buffer (BD Pharmingen). The parasitized cells (TER119+ GFP+) or unparasitized cells (TER119+ GFP-) had been analyzed for PS expression with flow cytometry.In vitro PS externalization with FasL trepFasL trep (000 ng/ml) was added to a Strep-Tactin microtiter plate, and incubated for 20 min at 37 . Sorted erythroid cells from PyNL FP-infected gld mice (2 105 cells/well) have been cultured for 4 hr at 37 inside the abovementioned plate. The PS was then surface stained with PE-conjugated annexin V in annexin V binding buffer. In some situations, cells had been Fc-blocked and stained with APC- or PE-Cy7conjugated anti-TER119 antibody and PE-Cy7-conjugated anti-MHC class I antibody, then analyzed with flow cytometry.In vivo depletion of macrophagesMacrophage depletion techniques have been previously described (Van Rooijen and Sanders, 1994; Ishida et al., 2013). Mice had been intravenously injected with clodronate (Sigma) liposome (C/L: 1.5 mg clodronate/300-l liposome suspension) three and 9 days following PyNL infection.Statistical analysisTwo sets of information (manage vs experimental group) had been compared and Mann hitney U-test was applied for statistical analysis. A p-value of p 0.05 was regarded as to become statistically substantial. Considerable differences in survival were tested having a log-rank test employing Kaplan eier survival curves.AcknowledgementsThis study was supported by Grants-in-Aid (24117504 to HH, 24790399, 26860276 to TI) and also the Strategic Fund for the Promotion of Science and Technologies to HH in the Ministry of Education, Culture, Sports, Science and Technologies of Japan; the Ministry of Wellness, Labour and Welfare of Japan (H24-Shitei-004) to HH; and also the Takeda Memorial Foundation to HH.Imai et al. eLife 2015;4:e04232. DOI: 10.7554/eLife.19 ofResearch articleImmunology | Microbiology and infectious diseaseAdditional informationFundingFunder Grant reference Author BRPF3 Inhibitor custom synthesis Hajime Hisaeda Takashi Imai Takashi Imai Hajime Hisaeda Hajime Hisaeda Hajime Hisaeda Japan Society for the Promotion Grants in Aid 24117504 of.