Flow cytometric analysis of CD4+ T lymphocytes in peripheral lymphoid tissuesafter L5Tx. WT BALB/c mice were subjected to either L5Tx or sham surgery. Splenocytes (from individual mice) and lumbar LN cells (pooled from 5 mice) were collected at 7 days after surgery and analyzed via intracellular flow cytometry with a combination of mAbs against CD45, CD3, CD4 and either T-bet, GATA-3, IFNγ, or IL-4 (see Methods). In A, representative images from a day 7 L5Tx spleen sample are shown to illustrate the analysis process. Total lymphocytes were first gated (R2 in the left panel), and CD4+ T lymphocytes (CD45+CD3+CD4+ population) were then selected for the analysis of each specific intracellular marker (middle and right panels, with T-bet used as an example): The final % of T-bet+ CD4+ T lymphocytes in one particular sample is the difference between the % of PE+(T-bet+) CD4+ T lymphocytes population obtained from T-bet-stained (CD45, CD3, CD4 and T-bet) sample (right panel) and the % of PE+ CD4+ T lymphocytes population obtained from the corresponding surface-stained only (CD45, CD3 and CD4) control (middle panel). In B and C, the final calculated % of T-bet+, GATA-3+, IFNγ+, or IL-4+ CD4+ lymphocytes within the total splenic lymphocytes (B, n = 7–10 mice per group) and the total lymphocytes from lumbar LNs (C, n = 3–4 repeats per group) are shown. All data are presented as mean ± SEM.