G6⁺ B cells are prone to apoptosis and cell death, and the apoptotic cells are disproportionately CD21^low. PBMCs were incubated for 6 hours in the presence of media alone, mouse IgG₁ (isotype control), or 1 μg/mL anti-CD95 with 2 μg/mL protein G. Cells were then stained with annexin V, 7-AAD, anti-CD20, anti-CD21, and G6 mAbs and were analyzed by flow cytometry. Analyses of G6⁺ and G6⁻ CD20⁺ B cells are shown. (A) Annexin V and 7-AAD staining of cells at baseline and after 6 hours of stimulation. (B) Analysis of surface CD21 and CD20 expression on cells incubated for 6 hours with α-CD95/protein G; annexin V⁺7-AAD⁺, annexin V⁺7-AAD⁻, and annexin V⁻7-AAD⁻ subsets. (C) Cell surface anti-CD20 (of total lymphocytes), G6 (of total B cells), and anti-CD21 (of G6⁺ and G6⁻ B cells) staining at baseline, compared with 6 hours, of stimulation. PBMCs from LDU 125 were collected 10 months after the cells collected for the microarray and primary immunophenotyping experiments. Data are representative of 3 independent experiments.