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.