Splenic DCs increase expression of CD200 on the cell surface as they undergo apoptosis. (A) Freshly isolated splenocytes from WT C57BL/6 or CD200^-/- mice were quadruple stained with CaspACE, anti-CD200 mAb, CD11c, and 7-AAD. Cells shown are 7-AAD^- and CD11c⁺. Numeric values denote the percentage of cells in each quadrant. (B) CD11c⁺ DCs were cultured in complete DMEM for 24 hours and assayed for cell size (FSC) versus CD200 expression. Quadrants are arbitrarily set on the basis of minimal (< 5%) staining with isotype control antibody. Numeric values denote the percentage of CD200⁺ cells. (C) CD11c⁺ cells were cultured in complete DMEM for 24 hours and sorted into either nonapoptotic (Annexin V^-/7-AAD^-) or apoptotic (Annexin V⁺/7AAD^-/+) populations, and CD200 expression was examined. (D) CD11c⁺ cells were cultured in complete DMEM for 18 hours and triple stained with CaspACE, 7-AAD, and anti-CD200 mAb. Electronic gates were set on nonapoptotic (CaspACE^-/7-AAD-), early apoptotic (CaspACE⁺/7-AAD^-), or late apoptotic (CaspACE⁺/7AAD⁺) cells, and CD200 expression was examined. (C-D) Shaded and unshaded histograms represent staining with anti-CD200 and isotype control mAbs, respectively. ΔG_mean values represent the change in MFI between anti-CD200 and isotype control staining. Results are representative of 3 replicate experiments.