Human cd3 cd28 beads

Isolated T cells were activated by the addition of human CD3/CD28 beads (Invitrogen, Carlsbad, CA, USA) to the cell culture medium at a ratio of 2 : 1 at 48 hours prior to transduction. Then, the engineered virus was added to the cell culture with a MOI of 10, followed by polybrene (Yeasen Biotech, China) at a final concentration of 5 μg/mL. Cells were centrifuged at 1200 × g for 60 minutes and incubated overnight at 37°C and 5% CO₂. At 5 days after the transfection, modified T cells were harvested and the expression of CAR was determined by flow cytometry and Western blot analysis.
All tumor cells, including MDA-MB-231, MDA-MB-468, and MCF-7 cells, were cultured to the log phase. When the cells reached a confluency of ∼70%, fresh complete medium containing 6 μg/mL polybrene and 50 μL of virus was added to each well of a 6-well plate. After incubation for 24 hours, the medium was replaced by 2 ml of fresh complete medium. After 5 days of transduction, cells expressing RFP were selected by medium containing 1.5 μg/ml puromycin (Beyotime, Shanghai, China). RFP was detected by flow cytometry and Western blot to determine whether the transfection was successful.