An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids
An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids
Transcrição
When the spheroids reach the appropriate experimental size, carefully angle the plate, and use a multichannel pipette to gently remove 150 microliters of complete medium from each well without disturbing the spheroids. Next, replace the discarded medium with 50 microliters of a 1-to-200 solution of Annexin-V red, and place the plate in a cell culture incubator for 15 minutes.
Centrifuge transduced CAR CD19 T cells in a 15-milliliter conical tube, and resuspend the pellet in 2 milliliters of complete medium after counting, dilute the cells to a 2 x 105 cells per milliliter of complete medium concentration.
Then, add 100 microliters of CAR CD19 T cells to each spheroid containing well, and return the plate to the automated imaging apparatus. In the acquisition software, select ScheduleparaAcquire and right-click on the ScanTimeline. Select EditTimeline, and right-click on the ScanGroup to delete it.
Right-click on the timeline again, and select SetSelectedScanGroupInterval, and SetAddScans Every to 1 and 1/2 hours and for a total of to 24 hours. Then set the imaging start time to at least one hour after the start of the incubation in the automated imaging apparatus, and select SaveScheduledScans.