Pe conjugated anti mouse cd4

The inguinal lymph nodes (LNs) from each treated group were prepared. Briefly, on experimental day 42, inguinal LN cells were extracted from each mouse using mechanical disruption with mesh, and single-cell suspensions were obtained. For intracellular detection of cytokines, LN cells (2 × 10⁶) were stimulated with CII (20 ug/ml) for 72 h; Golgi stop solution (BD Biosciences) was added for 4 h before the culture cells were harvested. The cells were then washed twice in FACScan buffer and stained with a phycoerythrin (PE)-conjugated anti-mouse CD4 (Biolegend) Ab. Cells were then fixed and processed for intracellular staining using the Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions. The FITC-conjugated mAbs specific to murine IFN-γ, IL-17A, and Foxp3 were purchased from BioLegend. All samples were detected using an Accuri 5 flow cytometer, and the mean fluorescence intensity was calculated using a C6 Accuri system software (Accuri Cytometer)

Cells were incubated with specific antibodies for 15 min at 4 °C in the dark for cell surface staining. For intracellular staining, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μg/ml ionomycin (MCE) in the presence of GolgiStop (BD Biosciences) for 4 h. Subsequently, cells were fixed and permeabilized with BD Cytofix/Cytoperm Plus (BD Biosciences), and incubated with specific antibodies for another 30 min at 4 °C in the dark. All samples were acquired with FACSVerse flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar). The following antibodies were used: PE-CY7-conjugated anti-mouse F4/80 (BioLegend), PerCP-conjugated anti-mouse CD11b (BioLegend), APC-CY7-conjugated anti-mouse CD45RA (BioLegend), PerCP-conjugated anti-mouse CD3 (BioLegend), PE-conjugated anti-mouse CD4 (BioLegend), APC-conjugated anti-mouse CD4 (BioLegend), Bv421-conjugated anti-mouse IL-17A (BioLegend), PE-conjugated anti-mouse IL-17A (BioLegend).