Flow cytometric analysis of CD4+ T lymphocytes in the lumbar spinal cord post-L5Tx. WT BALB/c mice were subjected to either L5Tx or sham surgery. Lumbar spinal cord mononuclear cells (pooled from 10 mice) were collected at 7 days after surgery and analyzed via intracellular flow cytometry with a combination of mAbs against CD45, CD3, CD4, and one of the following: T-bet, GATA-3, IFNγ, IL-4, TNFα, GM-CSF (see Methods). Experiments were repeated multiple times for statistical analyses. In A, representative images from lumbar spinal cord samples are shown to illustrate the analysis process. Total cell population was first gated (R1 in (a)), and then infiltrating leukocytes (CD45hi) were identified (b). CD4+ T lymphocytes (CD45+CD3+CD4+ population) (c) were further selected for the analysis of each specific intracellular marker ((d)–(f), T-bet is used as the example): The final % of T-bet+ CD4+ T lymphocytes in each sample is calculated as described in Figure 1A using “surface stained only” as control (d). Representative plots from a “Day 7 L5Tx” sample and a “Day 7 Sham” sample are shown in (e) and (f) respectively. Based on the total number of mononuclear cells collected, the number of T-bet+, GATA-3+, IFNγ+, IL-4+, TNFα+, and GM-CSF+ CD4+ T lymphocytes were calculated. In B–E, the calculated numbers of T-bet+ (B, n = 4), IFNγ+ (C, n = 5), TNFα+ (D, n = 6), and GM-CSF+ (E, n = 6) CD4+ lymphocytes per group of pooled lumbar spinal cords (10 mice) are shown. All data are presented as mean ± SEM. Each data point represents one pooled sample used. Note that, in some graphs, data points with similar values overlap. * indicates p < 0.05 between the indicated group and all other groups in the same graph.