2.7. Tumor cells apoptosis assay
CAR-T cells and target cells were incubated for 2h at 37°C, and target
cell apoptosis was assayed using an Annexin V/7-AAD Apoptosis Detection
Kit (Biolegend), according to the manufacturer’s instructions. Target
cell caspase-3 activity was detected used a caspase-3 activity detection
assay kit (BD). In brief, the cells were washed twice with cold PBS,
followed by fixing and permeabilizing in Cytofix/CytoPerm for 20 min on
ice (BD Biosciences). Subsequent staining was performed with 1×PermWash
as the staining and wash buffer and incubated for 15 min at room
temperature. Washed cells were analyzed with a BD celesta (BD
Biosciences) software by flow cytometry.