2.4 Flow cytometry analysis
PBMC was extracted from OME and CON rat spleen using rat lymphocyte separation solution (Solarbio Science, Beijing, China), and T cells were sorted by flow cytometry. To detect Treg cells, PBMC stained with eZFluorTM anti-human CD4-FITC and CD25-APC mixture, and incubated with an effective fixation/permeation solution. According to the manufacturer’s instructions (Thermo Fisher, Waltham, Massachusetts, US), intracellular staining was performed with anti-human FOXp3-PE or rat IgG2a K isotype control PE. To evaluate Th17 cells, PBMC was stimulated for 5 hours in a complete medium (supplemented with 10% FBS and 200 mM L-GLN RPMI 1640) and then incubated with FITC, an anti-human CD4-FITC, or mouse IgG1 K isotype control, and then with an effective fixative/permeable solution and permeable buffer. According to the manufacturer (Thermo Fisher, Waltham, Massachusetts, US), intracellular staining was performed with anti-human IL-17A-PE or mouse IgG1 K isotype control PE. FACSCalibur flow cytometry (BD Biosciences, US) was used for analysis and CellQuest software (BD Biosciences, Franklin Lakes, NJ, US) analyzed data.