Apc conjugated il 17a

To analyze the expression of cytokine of CD4⁺ T cells, cell surface staining was performed using pacific blue-conjugated CD4 (Biolegend) for 20 min at 4 °C in the dark. After the stained cells were washed with PBS, intracellular staining was performed to detect TNF-α, IFN-γ, IL-17A, and IL-4 expression. According to the manufacturer’s instructions, the cells were fixed and permeabilized using a Transcription Factor Staining Buffer Set kit (eBiosciences) and stained with allophycocyanin (APC)-cy7 conjugated TNF-α, (phycoerythrin) PE-cy7-conjugated IFN-γ, IL-4, or APC-conjugated IL-17A (eBiosciences). Prior to cytokine analysis, the purified CD4⁺ T cells were restimulated with 40 ng/mL phorbol-12-myristate-13-acetate (Sigma-Aldrich) and 1 µg/mL ionomycin (Sigma-Aldrich) for 5 h. Monensin (Sigma-Aldrich) was added at a concentration of 4 µM for the last 1 h of TCR stimulation or the last 6 h of PHA stimulation.