When doing T-cells activation assay, for example looking for CD25 ,CD69 expression or ELISA for cytokine release, is there a difference between the coated antibody or coated protein stimulation and cell stimulation using cell lines? What is guide to use the stimulus.?
Thanks,
Abdul
Short answer, yes there is. The first two is almost always global, with the last one... with the potential of being more specific.
For coated antibodies, it is almost always CD3/CD28, and the coating is for crosslinking. Otherwise, it doesn't work.
As for protein... do you mean an mitogen? Like ConA? or a cytokine like IFNg? Either way, the activation pathways are different and for these, there is no need to coat the plate. They will work in suspension.
Cell stimulation using cell lines...Unless you are looking at stimulation using unmatched HLAs...Most of this is using cell lines preloaded with a specific antigen or antigenic peptide.
If you can give more information about your goal... we can give you an idea of what would be the best way to go about it.
Shen-An Hwang Thanks for your reply
Sorry for not being very clear. I have T-cells transduced with CD19 CARs and looking into the different ways of activation. I meant by coated protein is to use an active form CD19 protein to evaluate the activation. Another way is with cell line such as Raji which expresses CD19. So there will be a difference in activation between the cell lines and the coated antibodies or protein.
So what I was looking for is there a guide or points to consider when choosing the way of activation?
Thanks
Depends on what you want to measure. To prove that your CD19 CAR works, I'm pretty sure it's suppose to induce targeted cell death.
Yes, you can use protein, and that in it self will stimulate T cell cytokines.
If you use the cell line, the read out is almost always cell death of the target cell. And the secondary read out is the cytokine profile.
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