Macs cd8 t cell isolation kit

To assess CD8⁺ T-cell proliferation in vivo, 200 μL of 5-bromodeoxyuridine (BrdU) labeling reagent (Life Technologies, Carlsbad, CA) was administered by i.p. injection. After 2 h, splenocytes were harvested, fixed and permeabilised using Nuclear Factor Fixation and Permeabilisation buffers (Biolegend). The samples were then labeled with anti-BrdU mAb (BU20A; eBioscience) and analyzed by cytofluorimetric analysis. For in vitro assays, splenocytes or purified CD8⁺ T cells were labeled with 2.5 μM of carboxyfluorescein succinimidyl ester (CFSE) and 3 d later CFSE dilution was measured by fluorescence cytometry. Proliferation was calculated as the percentage of CFSE low cells relative to the undivided CFSE-labeled immune cell peak fluorescence. For purification and transfer of total CD8⁺ T cells, a magnetic-activated cell sorting (MACS) CD8⁺ T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), was used to isolate an 'untouched' CD8⁺ T-cell population with > 90% purity. Purified cells were injected i.v. at equal numbers to groups of recipient mice 1 d prior to tumor challenge.

This study was approved by the Human Research Ethics Board of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital and conformed to the Declaration of Helsinki. Informed consent was obtained from participants. PBMCs were isolated from the blood of healthy donors by using Ficoll-Hypaque (Sigma, St. Louis, MO, USA) density gradient according to the manufacturer’s instructions. T cells were positively selected from PBMCs using the MACS CD8+ T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and activated using microbeads coated with anti-human CD3 and anti-human CD28 antibodies at a 1:2 bead/cell ratio. The cells were cultured at a density of 2 × 10⁶ cells/mL for 2 days in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA), 30 IU/mL recombinant human IL-2, 10 mM HEPES, 2 mM glutamine, and 1% penicillin-streptomycin.