Cd45 apc clone 30 f11

Splenocytes were stimulated using Cell Activation Cocktail (with Brefeldin A) (BioLegend) at 37 °C and 5% CO₂ for 5 h. Cells were then collected, and dead cells were stained with Zombie Aqua staining (BioLegend). The Intracellular Fixation & Permeabilization Buffer Set (eBioscience) was used for intracellular cytokine staining. Cells were fixed for 20 min at 37 °C. Permeabilized cells were then stained with IL-6-APC (clone RM4-4, dilution 1:200, BioLegend, Cat 116008). For Foxp3 staining to evaluate regulatory T cells, True-Nuclear Transcription Factor Buffer Set (BioLegend) was used. Briefly, splenocytes or draining lymph node lymphocytes were collected and fixed for 45 min, washed with permeabilization buffer, and stained with Alexa Fluor 488-conjugated anti-Foxp3 and isotype control antibodies (eBioscience), followed by surface staining with CD4-Pacific blue (clone RM4-4, dilution 1:200, Cat 116008), CD25-PE (clone 7D4, dilution 1:200, BD biosciences, Cat 558642), CD3ε -PE/Dazzle 594 (clone 145-2C11, dilution 1:100, Cat 100347) and CD45-APC (clone 30-F11, dilution 1:400, BioLegend, Cat 103112) at 4 °C for 30 min. Data were acquired using FACS LSR Fortessa (Becton, Dickinson and Inc.) and analyzed using the FlowJo Software 10.6.2 (BD Biosciences).