Cd3 dynabeads

Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors by Ficoll-Paque density centrifugation. Then, T cells were isolated from PBMCs using magnetic cell separation (MACS) and CD3 Dynabeads (Miltenyi Biotec, Germany) and cultured in X VIVO-15 medium (LONZA, Switzerland) supplemented with 200 IU/ml IL-2 and CD3/28 Dynabeads (Gibco, USA).
T cells were transduced by incubation with a CAR lentivirus (CAR-T) or vector lentivirus (Control T). After immediate centrifugation for 90 min at 37 °C, the transduced cells were cultured at 37 °C and 5% CO₂ for 48 h. LMP1-CAR-EGFP fusion vector was used for the detection of transfection efficiency and in vitro cytotoxicity assay. LMP1-CAR vector without EGFP was used for the detection of T cell activation markers, cytokine production and in vivo experiments. The transfection efficiency of CD38-CAR or two Tan CAR-T cells was detected by fluorescence-activated cell sorter (FACS) using fluorescein isothiocyanate (FITC)-labelled recombinant human CD38 protein (ACROBiosystems, China).

Informed consent was obtained before the study started, and the study was approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Lot No. 2022‐KY‐1062). T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using magnetic activated cell sorting (MACS) method with CD3 Dynabeads (Miltenyi Biotec). Then they were cultured and expanded in X VIVO‐15 medium (LONZA) supplemented with 200 IU/mL IL‐2 for 3–5 days. T cells were transduced by incubation of CAR lentivirus (CD38‐CAR‐T) or vector lentivirus (Control T). Forty‐eight hours later, transfection rate of T cells was detected by FACS with recombinant human CD38 protein (FITC‐labeled, ACROBiosystems).