This study investigates the cytotoxic effects of chimeric antigen receptor T cells (CAR T cells) on tumor spheroids. By utilizing fluorescent probes, the research aims to visualize apoptosis in cancer cells following CAR T cell treatment.
Take a multi-well round-bottom plate containing tumor spheroids, three-dimensional clusters of transduced cancer cells expressing a specific surface antigen, and a green fluorescent protein.
Carefully replace the media with Annexin-V red, a fluorescent apoptosis probe.
Now, introduce transduced chimeric antigen receptor T cells (CAR T cells) expressing chimeric antigen receptors to the wells.
These engineered receptors on CAR T cells recognize and bind to antigens on cancer cells within the spheroids, activating the CAR T cells.
This binding triggers the release of granules containing cytotoxic molecules, inducing cancer cell apoptosis.
In apoptotic cells, phosphatidylserine residues translocate to the cell membrane's outer leaflet.
Further, Annexin-V red probes with high affinity for phosphatidylserine bind to exposed residues, facilitating apoptotic cell detection.
Acquire time-lapse fluorescent images of the tumor spheroids following CAR T cell addition.
A decrease in green fluorescence from cancer cells in spheroids, coupled with an increase in red fluorescence indicative of apoptosis, indicates CAR T cell cytotoxicity against tumor spheroids.
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 Schedule to Acquire and right-click on the Scan Timeline. Select Edit Timeline, and right-click on the Scan Group to delete it.
Right-click on the timeline again, and select Set Selected Scan Group Interval, and Set Add Scans 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 Save Scheduled Scans.
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