Alexa fluor 647 labeled polyclonal goat anti mouse igg h l antibodies

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).

For each analyzed T cell culture, one sample of cells was stained with Alexa-Fluor 647-labeled polyclonal goat anti-mouse IgG (H+L) antibodies (Affinity) to detect CAR-T cells. In addition, we also detected the expression of the CAR with the QBEND-10 monoclonal CD34 antibody (Abcam) labeled with an RQR8 tag. Subsequently, all cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD3 antibodies (Abcam).