2.9. Flow cytometry analysis of Th1, Th2, Th17, Treg, and multifunctional T cells

Single-cell suspensions were seeded into U-shaped bottom microplates and incubated in the absence or presence of 1 × Cell Stimulation Cocktail for 4 h at 37 °C. After stimulation, the cell surfaces were stained with L/D, anti-CD3, anti-CD4, anti-CD8, and/or anti-CD25 mAbs for 30 min at 4 °C. The cells were then washed with cold PBS and centrifuged at 1500 × g for 3 min at 4 °C. The pelleted cells were resuspended in 100 μL of a Cytofix/Cytoperm solution (for Th1, Th2, Th17, multifunctional T cell analysis; BD Bioscience) or transcription factor staining buffer (for Treg cell analysis; ThermoFisher Scientific) per well for 30 min at 4 °C, and the cells were washed with 1 × Perm/Wash solution. Next, the cells were stained with antibody cocktails for Th1/Th2/Th17 cell detection (anti-IFN-γ, anti-IL-5, and anti-IL-17A mAbs), multifunctional T cell detection (anti-IFN-γ, anti-IL-2, and anti-TNF-α mAbs), or Treg cell detection (Foxp3 mAb) for 30 min at 4 °C. After intracellular staining, T cells were analyzed using a flow cytometer and the FlowJo software.