Effect of CY on the absolute and relative numbers of lymphocytes in spleens and LNs. (A) The effect of CY on spleen cell number. At various time points after the administration of low-dose (2 mg/mouse) CY intraperitoneally, total splenocytes were counted using a hemocytometer after lysis of red blood cells with ammonium chloride (ACK) buffer. Results are representative of 2 experiments. Results are presented as the mean ± SD of results from 3 mice in each of 2 experiments. (B) Effect of CY on spleen cell phenotype. At various time points after the administration of low-dose (2 mg/mouse) CY intraperitoneally, spleen cells were stained with fluorescence-labeled antibodies against lymphocyte markers. Relative numbers of CD4⁺(), CD8⁺(▪), and CD19⁺ cells (▴) in the spleens were determined by flow cytometry. (C) Effect of CY on LN phenotype. At various time points after the administration of low-dose (2 mg/mouse) CY intraperitoneally, LNs were stained with fluorescence-labeled antibodies against lymphocyte markers. Relative numbers of CD4⁺, CD8⁺, and CD19⁺cells in the LNs were determined by flow cytometry. Results are presented as mean ± SD of results from 3 mice. (D) Effect of CY on the percentage of CD4⁺cells coexpressing CD25. At various time points after the administration of low-dose (2 mg/mouse) CY intraperitoneally, spleens () and LNs (▪) were stained with fluorescence-labeled antibodies against T_REG markers. The percentage of CD4⁺ cells that express CD25 was measured using dual-color flow cytometry. Results are the mean ± SD average of triplicate stains for 2 different mice. (E) Effect of CY on the number of CD4⁺25⁺ T cells in the spleens. The number of CD4⁺25⁺ cells in the spleens at various times after CY administration was calculated [(spleen cell count) × (% CD4⁺) × (% CD25⁺ of CD4⁺)].