Figure 2.
Mouse peritoneal macrophages express P-selectin. Flow cytometry was performed on peritoneal cells freshly isolated from wild-type and P-selectin null mice. (A) Peritoneal leukocytes were labeled with a rat monoclonal anti–P-selectin antibody or with an isotype-matched rat IgG. Cells from wild-type mice labeled with anti–P-selectin antibody (shaded), cells from P-selectin null mice labeled with anti–P-selectin antibody (thin line); cells from wild-type mice labeled with isotype-matched rat IgG (thick line), cells from P-selectin null mice labeled with isotype-matched IgG (dashed line). (B) Peritoneal leukocytes were labeled simultaneously with FITC-conjugated rat monoclonal anti–P-selectin antibodies and with either PE-conjugated rat anti-F4/80 antibodies or PE-conjugated rat anti-CD2 antibodies. (C) Peritoneal cells labeled with FITC-conjugated anti–P-selectin antibodies and separated on a cell sorter according to P-selectin expression were stained with Wright-Giemsa stain and visualized by light microscopy. Left, unfractionated cells; center, P-selectin–positive cells; right, P-selectin–negative cells. (D) Proteins in the lysate from platelets (108) and peritoneal leukocytes (4 × 106) were separated by electrophoresis on 7% SDS-gels under reducing conditions and the proteins transferred to a PVDF membrane. P-Selectin was detected using rabbit antibodies to the cytoplasmic tail of P-selectin and HRP-conjugated goat anti–rabbit IgG. WT, wild-type mice; P−/−, P-selectin null mice; PSGL-1−/−, PSGL-1 null mice; hP, purified human P-selectin (0.1 μg); WEHI, lysate from 4 × 106 WEHI cells; Pl, platelet lysate; Le, peritoneal leukocyte lysate. (E) Total mRNA was isolated from peritoneal leukocytes of wild-type (WT) or P-selectin null (P−/−) mice, the mRNA species separated by gel electrophoresis and transferred to a PVDF membrane. The P-selectin band was detected using radiolabeled probe complementary to exon 3 of mouse P-selectin. (F) Peritoneal leukocytes were labeled simultaneously with FITC-conjugated rat monoclonal anti–P-selectin antibodies and with PE-conjugated rat anti–L-selectin antibodies, PE-conjugated anti–PSGL-1 antibodies, PE-conjugated anti-Mac1 antibodies or PE-conjugated rat anti–VLA-4 antibodies.