New to the field of cell therapy. Why fluorescent-labeled antibodies to target cell antigens are not used for determining target cell count by flow cytometry in cytotoxicity assay (eg car-t)? Cell dyes are always used to label target cells. Why?
Thank you in advance!
Hi Daniel,
The cells can be stained with intracellular 7aad and pi to quantify apoptosis of target cells. But more specifically, car t cells directed against b cell malignancies usually are designed to recognize cd20. I think an argument cam be made that it is better to label the target cells intracellularly so as not to impede conjugate formation between target cell and effector cell, and using for example anticd20 would clearly do that. Another reason is that the antibody can potentially bind to Fc receptors on the target cells. Allowing the antibody to crosslink target cells would cause a biased assay outcome.
Lora Benoit is right that the use of 7-AAD and PI are to stain dead cells. Surface antigens are not lost during the cytotoxic attack by the CAR-T cells, so you would not see killing, only total target cells.
Typically the chimeric antigen receptor is targeted to CD19, but some CARs are both CD19 and CD20 specific, to improve killing.
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