Cd3 cd28 stimulation beads

CD3⁺ T cells from healthy donors were separated from PBMCs using CD3 immunomagnetic beads (#130-097-043, Miltenyi Biotec, Germany), then amplified using CD3/CD28 stimulation beads (#11131D, Thermo Fisher Scientific) and IL-2 (100 IU/mL; Miltenyi Biotec) in X-VIVO 15 medium (Lonza). Cells were activated and expanded for 48 hr followed by transduction 2 hr later with lentivirus. T cells were generally engineered for 9–12 days to express a CD19-specific CAR and stained with Alexa-Fluor 647-labeled polyclonal goat anti-mouse IgG (H+L) antibodies (Affinity) to detect CAR-T cells. All cells were further confirmed by staining with fluorescein isothiocyanate (FITC)-labeled anti-CD3 antibodies (Abcam).

CD3+ T cells were separated from PBMCs using CD3 immunomagnetic beads (#130-097-043, Miltenyi Biotec, Germany) on day 1. T cells were amplified using CD3/CD28 stimulation beads (#11131D, Thermo Fisher Scientific) and IL-2(100 IU/mL; Miltenyi Biotec) in X-VIVO 15 Cell Medium (Lonza). Cells were then activated and expanded for 48 hours were transduced 2 hr later with the lentivirus (multiplicity of infection is 10) by different temperatures incubation in the presence of polybrene (Sigma) at 8 μg/mL. Then, the cells continue to expand at 37 degrees at an appropriate concentration (0.5-1×106 cells/ml). The transduction efficiency was determined 3 days after transduction. Generally, the T cells were engineered via 9-12 days of manufacturing to express a CD19-specific CAR or CD123-specific CAR.