FSAP binding and activation upon contact with apoptotic and necrotic cells. After incubation of r-plasma with apoptotic and living cells, cells were incubated with an anti-FSAP antibody. Cells were stained with a PE-labeled rabbit-anti-mouse F(ab')2 antibody and FSAP binding was measured with flow cytometry (1A). Cells being double positive for propidium iodide and Annexin V were considered to be apoptotic (not shown). After incubation of r-plasma with apoptotic (A), necrotic (N) and living (L) cells, FSAP was immunoprecipitated with anti-FSAP-4. R-plasma incubated with buffer (B) was used as a negative control. Samples were applied to SDS-PAGE 4 to 12% gel under reduced conditions and blotted onto a nitrocellulose membrane. Detection was performed by using biotinylated anti-FSAP-9 recognizing the light chain (1B). scFSAP, single chain FSAP; LC, light chain.