Fig. 8

The flow cytometry analysis of cell percentages in CD3⁻/CD4⁺/CD8⁺ and Th1/Th2 cytokine expression in CD4⁺ leukocytes. A Three-color staining was used to analyze the expression of CD3⁺ CD4⁺ (CD4⁺ T cells) and CD3⁺ CD4⁻ (CD8⁺ T cells) in the CD45⁺ cells. The lymphocytes were gated on an SSC (side scatter) vs. CD45⁺ cells dot plot as gate region-P1 (P1). Three separate populations of CD45⁺ cells appeared in this three-color staining. The CD3⁻ expression cells are on the bottom left. Expressions of CD3⁺ CD4⁺ cells (CD4⁺ T cells) on the upper right and CD3⁺ CD4⁻ (CD8⁺ T cells) on the bottom right. B Data are presented as the mean ± SD of three independent experiments. A total of 1 × 10⁴ cells were analyzed for each sample. #: p < 0.05 compared to the Naïve group; *: p < 0.05 compared to the saline group. C Three-color staining was used to analyze the expression of Th1-type cytokine (IFN-γ) and Th2- type cytokine (IL-4) in the CD4⁺. Lymphocytes were gated on an FSC (forward scatter) vs. SSC (side scatter) dot plot as gate region-1 (R1). Expression of IFN-γ in CD4⁺ T cells gated as region-2 (R2) and IL-4 in CD4⁺ T cells gated as region-3 (R3). D Percentages of IFN-γ⁺/CD4⁺ and IL-4⁺/CD4⁺ T cells after immunotherapy present as a histogram of means ± SD. A total of 1 × 10⁴ cells were analyzed for each sample. E The expression portion of IFN-γ⁺/CD4⁺ and IL-4⁺/CD4⁺ T cells is presented as a percentage. The ratio of Th2/Th1 was calculated as IL-4⁺/CD4⁺ divided by IFN-γ⁺/CD4⁺. #: p < 0.05 compared to the Naïve group; *: p < 0.05 compared to the saline group. F Cell viability assay with propidium iodide staining by flow cytometry. Fluorescence greater than 10⁴ is regarded as dead cells and presented as P1. G Percentage of cell viability presented as mean ± SD in three independent repetitions