Fig. 2

Lactic acid suppressed cellular redox system of T cells. T cells were cultured in media with or without lactic acid (20 mM). Cells were incubated with the green and orange probes that react with ROS and superoxide. To measure GSH, cells were incubated at 24 °C with thiol green dye. Finally, the cells were analyzed by flow cytometry. Based on the differences between the mean fluorescence intensity of lactic acid-treated and untreated cells, the production of ROS, superoxide, and GSH were determined. (A) Shows representative gating strategies for ROS, superoxide and GSH production in T cells. Cells were gated on a forward vs. side scatter dot plot. (B) Representative histograms displayed MFI of ROS, superoxide, and glutathione in untreated (blue line) and lactic acid-treated group (red line). Bar graphs show the production of ROS(C), Superoxide (D), and glutathione (E) in T cells. Data are presented as mean ± SD from a representative experiment (n = 3). An Independent T-test was used to examine the difference between the two groups. (F) Gene expression of the oxidant molecule NOX-gp91phox and the antioxidant molecules SOD1, SOD2, Nrf2, and CAT in lactic acid-treated and un-treated T cells were examined. Bar graphs show gene expression levels of (a) gp91phox, (b) CAT, (c) SOD1, (d) SOD2, and (e) Nrf2 in the lactic acid-treated T cells normalized to T-media group. (*P < 0.05, ***P < 0.001) GSH: glutathione, SOD: superoxide dismutase, CAT: catalase, MFI: mean fluorescence intensity, SD: standard deviation