Anti rat cd25 pe

Peripheral blood mononuclear cells (PBMCs) were prepared by using a peripheral blood lymphocyte separation kit for rats (TBD Science, LTS1083, 200 mL/kit). To detect Tregs (cell density: 1 × 10⁶ cells) were stained with anti-rat CD4 FITC OX35 (Ebioscience, Cat. No. 11–0040-81) and anti-Rat CD25 PE (Ebioscience, Cat. No. 12–0390-80), and incubated for 30 min at 4 °C in the dark according to the manufacturer’s protocol. After rupturing the membrane by using a Foxp3 staining buffer set (Ebioscience, Cat. No. 00–5523-00), the cells were then incubated with anti-mouse/rat Foxp3 PE-Cy7 (Bioscience, Cat. No. 25–5773-80). Rat IgG2a K isotype controls PE-cyanine (Ebioscience, Cat. No. 25–4321-81) were used to compensate and confirm antibody specificity [16 , 17 ]. The percentage of CD4⁺CD25⁺Foxp3⁺Tregs and CD4⁺T cells were counted by using a FACS Canto II (BD, USA), and the CD4⁺CD25⁺Foxp3⁺Tregs/CD4⁺T ratio was calculated.

The following monoclonal antibodies (mAbs) used: anti-rat-Nrp-PE, anti-mouse-PD-L1-PE (eBioscience, San Diego, CA), anti-mouse/rat CD28-PE (Santa Cruz, CA), anti-rat CD4-FITC, anti-rat CD25-PE, anti-rat Foxp3-PE-cyanine 5 (eBioscience, San Diego, CA, USA). After membrane and intracellular staining, cells were analyzed on FACS CantoII using the BD FACSDiva v8.0 software.

Cells were analyzed on a flow cytometer (BD Bioscience) according to a previously reported method [34 (link)]. For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD PharMingen). The following mAbs were included: anti-rat CD25-PE, anti-rat CD4-FITC, and anti-rat Foxp3-PE-cyanine 5 (eBioscience, San Diego, CA, USA). As to the Foxp3 antibody, an isotype-matched control was used to determine the background. Tregs were identified as CD4⁺CD25⁺Foxp3⁺ triple-positive cells and were expressed as percentages in SC.