Phycoerythrin pe anti cd4 antibody

Intracellular cytokine staining and flow cytometry were modified from our previous report [30 (link)]. Briefly, on day 42, single cell suspensions from inguinal lymph nodes (ILNs) were pulsed with 20 ng/mL PMA (Sigma-Aldrich, St. Louis, MO, USA) and 1 μg/mL ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 18 h, with 10 μg/mL brefeldin A was added during the last 4 h of culture. After stimulation, the cells were surface stained with phycoerythrin- (PE-) anti-CD4 antibody (BD Biosciences, San Diego, CA, USA), permeabilized/fixed with cytofix/Cytoperm Plus (BD Biosciences), and stained with FITC-anti-IL-17A antibody and FITC-anti-IFN-γ antibody (Biolegend). To analyze regulatory T cells (Tregs), single cell suspensions from ILNs were stained with PE-anti-CD4 antibody (BD Biosciences), fixed, permeabilized, and stained with anti-Foxp3 antibody (BD Biosciences) according to the manufacturer’s instructions. Flow cytometer analysis was performed in an AccuriTM C5 cytometer.

Cell suspensions were prepared from lymph nodes obtained in sacrificed mice after 42 days. Cells (3 × 10⁵) were seeded in a 60-mm dish and cultured with Con A (5 µg/mL) for 24 h. During the last 4 h of culture, 10 μg/mL brefeldin A was added. Afterwards, cells were trypsinized and washed in phosphate buffered saline (PBS), and cells so collected were stained with phycoerythrin- (PE-) anti-CD4 antibody for 45 min (BD Biosciences, San Diego, CA, USA), washed twice with 0.1 BSA in PBS. Cells were then permeabilized and fixed with cytofix/Cytoperm Plus solution (#51-2090KZ, BD Biosciences, San Diego, CA, USA) for 20 min. Finally, cells were stained with FITC-anti-IL-17A (#506907, Biolegend, San Diego, CA, USA), FITC-anti-IFN-γ (# RM9001, eBioscience, Waltham, MA, USA), and with anti-Foxp3 (#560407, eBioscience, Waltham, MA, USA). Cells were analyzed with the AccuriTM C5 cytometer (Accuri Cytometers, Ann Arbor, MI, USA) and data processed with the BD Accuri™ C6 Plus software.