*3.5. Flow Cytometry Analysis*

To stimulate the production of lineage-specific markers, differentiated Th cells were treated with 50 ng/mL of PMA (Sigma) and 1 μM of Ionomycin (Sigma) with transport inhibitor GolgiStop (BD Biosciences) for 4 h. Cells were washed with PBS 4 times then incubated with FITC conjugated anti-human CD4 (BD Bioscience) at 4 ◦C for 30 min in the dark. After surface marker staining, cells were fixed and permeabilized using fixation and permeabilization buffer (BD Bioscience) then further stained with PerCP-Cy5.5-conjugated anti-human IFN-γ (BD Bioscience), APC-conjugated anti-human IL-4 (BD Bioscience), and PerCP-Cy5.5-conjugated anti-human FoxP3 (BD Bioscience) at 4 ◦C for 30 min in the dark. Nonspecific isotype-matched antibodies served as controls. Samples were analyzed with the BD FACS Verse flow cytometer (BD Biosciences) and data analysis was performed using FlowJo software.
