Cell therapy systems dynabeads cd3 cd28

GM-CSF^WT CART19 or GM-CSF^KO CART19 cells were stimulated with PMA/Ionomycin (Millipore Sigma, Ontario, Canada), CD19⁺ cell line Nalm6, or Cell Therapy Systems Dynabeads CD3/CD28 (Life Technologies, Oslo, Norway) at different time points (0hr, 1hr, 2hr, 4hr, 6hr, 1 day, 2 days, 3 days, 4 days, or 5 days). The following amounts were used for stimulation: 50 ng/mL of PMA, 1 ug/mL of ionomycin, 3:1 ratio of beads:cells, and 1:1 ratio of Nalm6:CART19. Then, cells were spun and washed with flow buffer, followed by incubation in the dark with the following reagents: CD3 (clone SK7) APC-Cy7 (344818, BioLegend, San Diego, CA, USA), Annexin V PE (556421, BD Biosciences, San Jose, CA, USA), 7-AAD (559925, BD Biosciences, San Jose, CA), and 1X annexin binding buffer (1:10 dilution of 10X ABB) (556454, BD Biosciences, San Jose, CA, USA). Then, the expression of Annexin V and 7-AAD on CD3 cells was measured via flow cytometry. For assays including recombinant exogenous hrGM-CSF (78190, Stemcell Technologies, Vancouver, Canada), the following doses were added: 100 and 1000 ng/mL. For exogenous FasL protein (FAL-H5241, ACRO Biosystems, Newark, DE, USA), the following dose was used: 50ng/mL.