Cell preparation and flow cytometric analysis for Th1/Th2/Treg/Th17 cells

Spleen tissues were collected, cut into small fragments, and teased apart to allow dispersion of the cells into RPMI 1640. The cells were passed through a 40 μm mesh to obtain a single cell suspension. Following a rinse, the cells were adjusted maximally to 2 × 10⁶ cells/ml. For Th1, Th2 and Th17 detection, the cells were activated with phorbol myristate acetate (PMA, 50 ng/ml; Alexis Biochemicals, San Diego, CA) and ionomycin(1 μM; Sigma, USA) in the presence of monensin (1 μl/ml; BD, USA) for 4 h at 37 °C in a 5% CO₂ atmosphere. For the analysis of Treg, cells were aliquoted into tubes for further staining.

We defined Th1 as CD3⁺CD4⁺IFN-γ⁺ cells, Th2 as CD3⁺CD4⁺IL-4⁺ cells, Treg as CD4⁺CD25⁺Foxp3⁺ cells, and Th17 cells as CD3⁺CD4⁺IL-17⁺ cells. The cells were incubated with PE-conjugated anti-rat CD3 and FITC-conjugated anti-rat CD4 for Th1, Th2, and Th17 analysis. The cells were incubated with FITC-conjugated anti-rat CD4 and PE-conjugated anti-rat CD25 for Treg analysis. After surface staining, cells were re-suspended in a fixation and permeabilization solution according to the manufacturer’s instructions and then stained with Anti-rat IFN-gamma eFluor® 660/Anti-rat IL-4 eFluor® 660/Anti-rat Foxp3 APC/Anti-rat IL-17A APC. All of the antibodies were from eBioscience. Fluorescence profiles were analysed using a FACScan cytometer equipped with CellQuest software (BD). The results are expressed as a percentage of positive cells.