Hi Everyone,
I did numerous human T cell CFSE proliferation assays, but the peaks never shift. I put CD3/CD28 and IL2, and I can see that T cells did proliferate by my eyes (as there was increase amount of T cells compared with several days before). I don't know why my CFSE never work. So frustrated. Is there any way or any other way to do human T cell proliferation assay?
Dear Suipwksiow!
Please You look at this protocol:
https://www.creative-biolabs.com/blog/index.php/the-introduction-of-t-cell-proliferation-assay/?gclid=EAIaIQobChMIl-CCxY7D7QIVJAZ7Ch3buglMEAMYAyAAEgLU3vD_BwE
T cell proliferation assay,
There are many articles in the literature on this. Check the protocols and make sure that everything is properly titrated.
Hello Suipwksiow Wjkp
1) Cells in culture are given a specific stimulus. For T cell, this is commonly plate-bound anti-CD3 antibody in combination with soluble anti-CD8 antibody. Other commonly used compounds to stimulate T-cell proliferation are Con A, PHA, as well as PMA and ionomycin.
2) A radio-labelled nucleotide is then added to the culture media. The most commonly used is 3H-thymidine. As the cells are stimulated to divide, they utilise the radio-labelled nucleotide in the culture media and incorporate it into their newly-synthesized DNA. Each successive generation of daughter cells will incorporate more of the radio-labelled nucleotide into its DNA.
3) After this incubation (generally 24-48 hours), the cells are removed from the culture media by centrifugation and washed to remove any free radio-labelled nucleotide that has not been incorporated into the cells’ DNA. The total radioactivity is measured, and compared against a control group of cells that did not receive the proliferation-inducing stimulus.
Good luck with your work.
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