Pe conjugated anti cd25 mab

The expression of the cellular activation markers CD69 (early), CD25 (late) and HLA-DR (very late) were measured on freshly isolated PBMCs after 3 days of incubation at 37°C with varying concentrations of LA, PRO2000, tenofovir and PHA. Briefly, the cells were washed with PBS/FCS2% and incubated with PerCP-conjugated anti-CD4 mAb (BD Biosciences) together with PE-conjugated anti-CD25 mAb (BD Biosciences), anti-CD69 mAb (Biolegend) or anti-HLA-DR mAb (BD Biosciences) for 30 min. at 4°C. In parallel, the cells were stained with SimulTest control IgGγ1-FITC/IgGγ2a-PE (BD) for aspecific background staining. After washing, the cells were fixed with 1% paraformaldehyde solution and analyzed with the FACSCalibur and Cell Quest software (BD).

The study protocol was approved by the medical ethics committee of affiliation hospital of Gansu University of Chinese Medicine and the informed written consents were obtained from all volunteers. Peripheral blood was obtained from healthy volunteers and the peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation over Ficoll (Histopaque 1077, Sigma Aldrich, USA). CD4⁺ T cells were enriched with an isolation kit (CD4⁺ T Cell Isolation Kit human, Miltenyi Biotec, Auburn, CA, USA), which contains a cocktail of CD8a, CD14, CD15, CD16, CD19, CD36, CD56, CD123, TcRγ/δ, and CD235a (Glycophorin A) antibodies. The cell separation was performed with LS columns (Miltenyi Biotech), according to the manufacturer's instructions. The CD4⁺ cells were further labeled with PE-conjugated anti-CD25 mAb (BD Bioscience, CA 555432, USA), PerCP-conjugated anti-CD4 mAb (BD Bioscience, 566321) and FITC-conjugated anti-CD127 mAb (BD Bioscience, 560549). The CD4⁺CD25⁺ Treg cells were obtained by cell sorting (purity > 95%) on MoFlo XDP (Beckman coulter, Pasadena, CA, USA). CD4⁺ cells and CD4⁺CD25⁺ cells were stimulated with anti-CD3/CD28-coated beads (Gibco, 11161D) (1:4), IL-2 (Gibco, PHC0026) (20 U/mL) in AIM-V (Gibco, 12055083) serum-free medium in 12-well plates (BD Labware).

For cell cycle assay, cells were first exposed to IR at a proper dose. Twenty-four hours post-irradiation, the cells were then fixed in -20°C prechilled 70% alcohol overnight, and stained with 20 μg/mL propidium iodide (PI) at room temperature. For apoptosis assay, cells were harvested 24 h post-irradiation and stained with Annexin V-FITC/PI (BD Biosciences, 556547) for 30 min at room temperature. For CD4⁺CD25⁺ Treg percentage assay, cells were labeled with PE-conjugated anti-CD25 mAb and FITC-conjugated anti-CD4 mAb (BD Bioscience, 550628).
Flow cytometry assay was performed using an Amnis imaging flow cytometer (Merck Millipore, Darmstadt, Hesse, Germany) and at least 10,000 gated events were acquired from each sample. The data were analyzed with IDEAS Application v6.0 (Amnis) or FlowJo v6.0 (Tree Star, Ashland, OR, USA).