Anti humancd25 pe cy7

Spleen tissue biopsy specimens from patients with SMZL and reactive spleen (rSP) were gently minced over a wire mesh screen to obtain a cell suspension. The cell suspension or peripheral blood from patients or healthy donors was centrifuged over Ficoll Hypaque at 1,200 rpm for 20 minutes to isolate mononuclear cells. CD3⁺T and CD8⁺T cells were isolated using negative selection with Human T-cell Enrichment Kit and Human CD8⁺T cell enrichment Kit, respectively (StemCell Technologies). Regulatory T-cell subsets (CD4⁺CD25⁺CD127^low) were isolated using a flow sorter after being stained with the following fluorochrome-conjugated antibodies: antihumanCD4-FITC (BD Bioscience), anti-humanCD25-PE-Cy7 (BD Bioscience), anti-humanCD127-BV421 (BD Bioscience).

For surface marker detection, 1 × 10⁶ or 1 million cells were washed in PBS and incubated with fluorochrome-conjugated antibodies (Abs) against and analyzed on a flow cytometer: anti-humanCD4-FITC (BD Bioscience), anti-humanCD25- PE-Cy7 (BD Bioscience), anti-humanCD127- BV421 (BD Bioscience), anti-humanCD161-APC (BioLegend), anti-humanCD26-PerCP/Cyanine5.5 (BioLegend). For apoptosis detection, Treg subsets were cultured in 96-well plate at 37°C in the presence of 5% CO₂. After 72 hours, cells were stained with PI/annexin V-APC and analyzed by flow cytometry. For Ki67 expression detection, Treg subsets were cultured in 96-well plate at 37°C and 5% CO₂ in the presence of Dynabeads Human T-Activator CD3/CD28 (Gibco). After 72 hours, cells were fix and permeabilized with reagents from Fixation/Permeabilization Solution Kit (BD Bioscience). Cells were then stained with anti-humanKi67-BV421 (BD Horizon) and analyzed by flow cytometry. See Supplementary Tables S4 and S5 for details of the flow cytometry antibodies and other reagents used.