Characterization of bone marrow-derived macrophages from PLSCR1 kwock-out mice and their phagocytotic activity.

<p><b>(</b>A) Histogram of CD11b expression by cells in BMDM cultures obtained from flow cytometric analysis of viable cells labelled with V450-anti-CD11b antibodies. Control sample was unstained. Nearly all cells (97%) in both PLSCR1 +/+ and PLSCR1 -/- cultures expressed the macrophage marker CD11b with similar MIFs, 6.7 and 7.0 respectively. (B) Confocal images of BMDM cultures prepared from PLSCR1 +/+ and PLSCR1 -/- mice. Cells were fixed and stained for F-actin with TRITC-phalloidin and macrophages were immunolabeled for Iba1 and visualized with a secondary antibody conjugated to Alexa 647. Bars, 50 μm. (C) Western blot of cell lysates (20 μg of total protein) of BMDM cultures from PLSCR1 +/+ and PLSCR1 -/- mice confirm the absence of PLSCR1 in knock-out mice. Blots were revealed with antibodies against PLSCR1, Iba1 and β-actin as loading control. (D) BMDMs from PLSCR1 +/+ (grey bars) or -/- (black bars) mice were incubated for 5, 10 or 30 min at 37°C with IgG-opsonized SRBCs, stained with Alexa488-anti-Rabbit IgG to detect external SRBCs before permeabilization and fixation for labeling of all associated SRBCs including internalized particles with Alexa647-anti-Rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs were counted for at least 50 cells. Results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured after 30 min of phagocytosis in BMDMs from PLSCR1 +/+ mice. Statistical significance was determined by the non parametric statistical hypothesis using the Mann-Whitney U test (n.s., p>0.05; ***, p<0.001).