Cell Staining for Flow Cytometry

For analysis of T helper cell lineages, live cells were stained with anti-CD3, anti-TCRβ, anti-CD4, and anti-MHCII with control IgG and Fc block. To distinguish transferred 7B8 T cells from the host cells, live cells were stained with anti-TCRVβ14, anti-CD45.1, and anti-CD45.2. Live-dead labeling with DAPI (for live cell analysis) was performed and only live cells were analyzed. For intracellular protein staining, extracellular proteins were first labeled with antibodies (anti-CD3, anti-TCRβ, anti-CD4, and anti-MHC-II), control IgG, and Fc-blocker for 30 min at 4 ^oC. After the cells were stained with live-dead labeling Aqua (Thermo Fisher Scientific), they were fixed using fixation/permeabilization buffer (Thermo Fisher Scientific) at room temperature for 30 min or at 4 ^oC for over night, re-suspended in permeabilization buffer (Thermo Fisher Scientific) and further stained with anti-Foxp3 and anti-RORγt using the Foxp3 staining buffer set (Thermo Fisher Scientific). To detect both cytokines and transcription factors together, one million LP mononuclear cells were stimulated with 50 ng/ml phorbol myristate acetate (PMA) (Sigma) and 500 ng/ml Ionomycin, in the presence of GolgiPlug (BD Biosciences) at 37 ^oC for 4 hours in a 5% CO₂ incubator. The cells were then incubated with control IgG and Fc-blocker for 15 min at 4 ^oC, and stained with anti-TCRβ, anti-CD4, anti-CD8a, anti-MHC-II for 30 min at 4 ^oC. Dead cells were further stained with Aqua. Following with the fixation and permeabilization, as described above, cells were stained with intracellular antibodies (Anti-IL-17A, anti-IL-22, anti-TNFα, anti-RORγt, and anti-Foxp3) for 30 min at room temperature. Non-specific or Fc-receptor binding were blocked with control IgG and Fc-blocker during staining. Flow cytometric analysis was performed on a LSRII (BD Biosciences), a FACS Aria (BD Biosciences), or Attune (Thermo Fisher Scientific). All data were re-analyzed using FlowJo (Tree Star).