Human pan t cells isolation kit

Peripheral blood samples were obtained from healthy donors after informed consent and approval was obtained by the ethical committee of the hospital according to the Helsinki declaration. PBMCs were isolated using Ficoll gradient (Sigma-Aldrich, St Louis, MO). CD19⁺ B cells and CD3⁺ T cells were purified by negative selection using the B cell isolation Kit II (Miltenyi Biotec) for CD19⁺ B cells and the human Pan T cells isolation kit (Miltenyi Biotec) for the CD3⁺ T cells following manufacturer's instructions followed by separation through the Automacs pro-separator (Miltenyi Biotec). Purity of isolated B and T cells was monitored using anti-hCD19APC and anti-hCD3PE antibodies (Miltenyi Biotec), respectively, and was analyzed by flow cytometry (MACSQuant VYB, Milteny Biotech).

Peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers and T cells were isolated from PBMCs by negative magnetic depletion using biotin-conjugated CD14, CD15, CD16, CD19, CD34, CD56, CD123, and CD235a following the manufacturer’s instructions of Human Pan T Cells Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Ficoll–Hypaque density gradient centrifugation was used to separate the PBMCs at 3000 rpm for 15 min under the manufacturer’s protocol. Flow cytometry was carried out to assess the purity of Tem cells to be more than 95% followed by maintaining in RPMI (Roswell Park Memorial Institute) 1640 medium (Thermo, Waltham, MA, USA) containing 10% FBS (fetal bovine serum) (Thermo, Waltham, MA, USA), streptomycin (100 μg/mL) and penicillin (100 U/mL) in 5% CO₂ at 37 °C for 1 h.