Figure 6. Real-time PCR analysis of RNA encoding Bcl-2 and Bax in CD4⁺ NKT, CD4-CD8⁻ NKT and NK1.1⁻ T cells and intracellular staining for IL-4 and Bcl-2 before and after irradiation.

(A) Fresh splenic NK1.1⁺TCRαβ⁺ T cells and NK1.1⁻TCRαβ⁺ T cells were sorted by flow cytometry from untreated mice, and from mice 10 minutes or 4 hours after a single dose of 1,000 cGy TBI. Total RNA was extracted from the cells and analyzed for expression of Bax and Bcl-2 by quantitative real-time PCR. Induction ratio of PCR products using primers specific for Bax and Bcl-2 were calculated from sorted cells in untreated mice versus irradiated mice at the two time points after irradiation. Mean ratio is shown for three independent experiments. Error bars represent SD. Dotted line shows threshold of changes from untreated mice. (B) The same analysis was performed for expression of Bax and Bcl-2 at 4 hours after irradiation using sorted CD4⁺NKT1.1⁺ and CD4⁻ NK1.1⁺ TCRαβ⁺ T cells pooled from the spleens of 10 mice. (C) Two color analysis of intracellular staining for IL-4, IFN-γ and Bcl-2 on gated CD4⁺ and CD4⁻ NKT cells before and 24 hours after irradiation with 10 doses of TLI. Representative flow cytometric patterns are shown.