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%.