Anti cd4 alexa488

Draining lymph nodes (LNs) were separated into single cells on postimmunization day 21 through a 70 µm cell strainer, and 5 × 10⁵ single cells, pretreated with stimulation Cocktail (plus protein transport inhibitors, Cat# 00-4975-93, eBioscience) for 6 hours, were dispensed into each tube along with intracellular/intranuclear staining fixation solution in 0.5% BSA. The antibodies staining was incubated with anti–IFN-gamma PE (Cat# 505807, BioLegend), anti–IL-17 Alexa647 (Cat# 506912, BioLegend), anti-CD4 Alexa488 (Cat# 100529, BioLegend), anti-CD25 PE (Cat# 12-0251-82, ThermoFisher), and anti-Foxp3 PE-Cy7 (Cat# 25-5773-80, eBioscience, San Diego, CA, USA). The stained cells were acquired on BD FACSMelody (BD Biosciences) and analyzed with FlowJo Software 10.5.3 (FlowJo LLC).

CB was from Merck-Millipore, Darmstadt, Germany. Anti-CD3 mAb, anti-CD28 mAb, anti-phosphotyrosine (anti-pTyr) PY20-Alexa 488, anti-CD4-Alexa 488, anti-CD25-PercP, anti-FOXP3-Alexa 488, and anti-TIM-1-PE were from Biolegend, San Diego, CA, United States. Rabbit anti-mouse-IgG1 and IgG2a were from Fisher Biotec, Hampton, New Hampshire, United States. Carboxyfluorescein diacetate succimidylester (CFSE) was from Biolegend. The 7-Plex T-Cell Receptor Th17 and IL-17 Magnetic beads MAGPIX Kits were from Merck-Millipore. Treg (CD4+CD25+CD127^low) and CD4+ T cell isolation kit were from Miltenyi Biotec, Bergisch Gladbach, Germany. The primers were from IDT, Coralville, Iowa, United States. The DNA Extraction and Purification Kit and Gel and PCR Clean-up System were from Promega, Madison, Wisconsin, United States. Recombinant Taq DNA Polymerase was from Thermo Fisher Scientific, Waltham, Massachusetts, United States, and QIA Quick PCR purification kit was from Qiagen, Hilden, Germany.