Rat anti mouse cd4 fitc

The purified spleen and liver cells were counted and their viability assessed using the trypan blue exclusion method.²⁹ Then, the absolute cell counts (2 × 10⁶) and suspension were in the complete RPMI 1640 medium. All cells were detected by BD FACSVerse and analysed by FlowJo software (Treestar, Inc, San Carlos, USA).
For Th1/Th2/Th17 analysis, the cells were stimulated for 4 h with 25 ng/mL PMA (Sigma‐Aldrich), 1 mg/mL Ionomycin (Sigma‐Aldrich) and 0.66 μL/mL Golgistop (Sigma‐Aldrich) and cultured at 37°C in 5% CO₂. The cells were harvested and stained with rat anti‐mouse CD3‐PerCP‐Cy™5.5 (BD Pharmingen, San Diego, USA) and rat anti‐mouse CD4‐FITC (eBioscience, San Diego, USA). After being fixed and permeabilized with FIX&PERM Kit (MultiSciences, Hangzhou, China), the cells were intracellularly stained with rat anti‐mouse IFN‐γ‐PE (eBioscience), rat anti‐mouse IL‐4‐PE (BD Pharmingen) and rat anti‐mouse IL‐17A‐PE (BD Pharmingen).
For Tregs analysis, single‐cell suspension was stained with rat anti‐mouse CD4‐FITC (eBioscience) and rat anti‐mouse CD25‐APC (BD Pharmingen). After being washed, fixed and permeabilized with Fixation/Permeabilization Diluent (eBioscience), then the rat anti‐mouse Fc block (BD Pharmingen) was added into cell suspension and incubated for 15 minutes at 4°C. The cells were intracellularly stained with rat anti‐mouse Foxp3‐PE (BD Pharmingen).

Th17 and Treg cells were detected with flow cytometry. Fresh spleen tissue was obtained under sterile condition, and single lymphocyte suspension (2–5 × 10⁶ cells/mL) was prepared. After treatment with stimulating or inhibiting agents, these cells were incubated with antibodies against Th17 and Treg cells (rat anti-mouse CD4-FITC, IL17A-PE, CD25-APC, and Foxp3-PE; all from eBioscience), respectively. Fluorescence was detected with a flow cytometer (FACSCalibur; BD, Franklin Lakes, NJ, USA).