2.5. Killing activity assay
The ability of CAR-T cells to kill tumor cells was determined via Calcein AM (CAM, Dojindo) release-based cytotoxic cell assay [26]. Briefly, target cells were stained with 10μM CAM for 30 minutes at 37℃, followed by terminating the reaction with addition of Durbe phosphate buffered saline without calcium (DPBS) buffer. The target tumor cells were washed five times with DPBS. Non-transduced T cells were used to normalize the percentage of CAR-positive cells. Then, CAR-T cells and target cells were plated in a 96-well microplate at various effector/target (E:T) ratios. After incubation for 6h at 37 °C, the 50ul supernatant were transferred to 96-black plates to measure fluorescence intensity (FI) at 485 nm excitation and 520 nm emission wavelengths. The percentage of cytotxicity was calculated according to the following formula: lysis% = (test release–spontaneous release)/(maximal release–spontaneous release)×100%.
The ability of CAR-T cells to kill 293T cells was detected via Real Time Cell Analysis (RTCA, ACEA). One prior day to seeded approximately 1*104 293T cells on 16 E-plate, the cells were grown to mid-logarithmic growth phase. Non-transduced T cells were used to normalize the percentage of CAR-positive cells, and CAR-T cells were added to the cultures at indicated effector-to-target ratio. RTCA DP analyzer was used to monitor real-time target cell growth, and the results were analyzed by RTCA software [27]. The percentage of cytotxicity was calculated according to the following formula: lysis% = [1 – (experiments/empty culture)] ×100%.