Effect of CY on apoptosis in CD4⁺25⁺T_REGs and CD4⁺25^- cells. (A-B) TUNEL staining of CD4⁺25⁺ cells. Two days after the intraperitoneal administration of 2 mg CY, spleen cells were removed, and CD4⁺25⁺ cells were separated by magnetic bead separation and fluorescence-activated cell sorting (FACS). CD4⁺25⁺ cells from treated or control mice were incubated with bound anti-CD3, and TUNEL staining was performed after 24 hours in culture. (A) CD4⁺25⁺ cells from untreated control mice. (B) CD4⁺25⁺ cells from CY-treated mice. Data are representative of 2 separate experiments. (C-D) TUNEL staining of CD4⁺25^- cells. Two days after the intraperitoneal administration of 2 mg CY, spleen cells were removed, and CD4⁺25^- cells were separated by magnetic bead separation and FACS. CD4⁺25^- cells from treated or control mice were incubated with bound anti-CD3 and after 24 hours in culture, TUNEL staining was performed. (C) CD4⁺25^- cells from untreated control mice. (D) CD4⁺25^- cells from CY-treated mice. Data are representative of 2 separate experiments. Percentages in panels A-D indicate the percent of apoptotic cells. (E-F) Annexin V staining of CD4⁺25⁺ cells. Two days after the intraperitoneal administration of 2 mg CY, spleen and LN cells were removed. Freshly isolated cells were stained for cell surface markers, and Annexin V staining was performed on the samples. (E) Annexin V profile of CD4⁺25⁺ LN cells from CY-treated mice (black solid histogram) compared with CD4⁺25⁺ LN cells from control mice (gray line histogram). (F) Annexin V profile of CD4⁺25⁺ spleen cells from CY-treated mice (black solid histogram) compared with CD4⁺25⁺ spleen cells from control mice (gray line histogram). (G-H) Annexin V staining of CD4⁺25^- cells. Two days after the intraperitoneal administration of 2 mg CY, spleen and LN cells were removed. Freshly isolated cells were stained for cell surface markers, and Annexin V staining was performed on the samples. (G) Annexin V profile of CD4⁺25^- LN cells from CY-treated mice (black solid histogram) compared with CD4⁺25^- LN cells from control mice (gray line histogram). (H) Annexin V profile of CD4⁺25^- spleen cells from CY-treated mice (black solid histogram) compared with CD4⁺25^- spleen cells from control mice (gray line histogram). Horizontal bars in panels E and H are gates indicating what percent of cells are positive for apoptotic cells.