Evaluation of apoptotic responses of PECAM-1^+/+ and PECAM-1^−/− splenic B cells upon IgM cross-linking and in vivo transfer of CFSE-labeled lymphocytes.

(A) T-cell–depleted splenic B cells were purified through a Percoll gradient and cultured in the presence or absence of whole antimouse IgM antibody. Cells were washed and stained with annexin V and propidium iodide at 0 and 17 hours after purification and the percentage of apoptotic cells determined on an EPICS XL-MLC flow cytometer. (B) A 2-dimensional dot plot of splenic lymphocytes taken from an individual mouse injected 7 days previously with CFSE-labeled PECAM-1^+/+ and PECAM-1 knock-out (−/−), which were “fully” or “one-quarter” labeled, respectively. In this example, simultaneous staining with the B-cell–specific antibody CD45R-PE allows calculation of the ratio of PECAM-1^−/−compared with PECAM-1^+/+ B cells. Similarly, CD5 staining was used to ratio T cells (data not shown). In addition, anti–PECAM-1 390 was used to confirm the identity of PECAM-1^+/+ and PECAM-1^−/− lymphocytes after transfer (data not shown). (C,D) Flow cytometric analysis allowed the calculation of the ratio of PECAM-1^−/− to PECAM-1^+/+ B and T cells prior to injection and at 7 and 30 days after transfer, demonstrating a decrease in PECAM-1^−/− B cells but not T cells.