Hi,

You can try to use almar blue to check proliferation of T cells. It works in a similar way to H3 incorporation but instead of radioactivity you measure color in a ELISA reader. I think you can find protocols online.

Thanks Mohanraj.

I find alamar blue gives a high background. I have tried it with a couple of assays. It works well to get cytotoxicity of a compound but I was not very happy with the proliferation assays.

Dear Lakshmi,

As Benjamin said, CFSE labeling is the most easy, practical, non-radioactive way of following proliferation. However, to my experience, quite a lot of optimization needs to be done to have clear signal. An alternative might be BrdU (Bromo-2'-deoxyuridine) labeling, much more expensive but very clean signal. However this method typically sees the divisions during the last hours of an experiment while CFSE can follow all divisions...

Kindest regards,

Ben

I dont want to do Flow based assay. since the number of samples are high.

Is ELISPOT a good assay to compare samples for antigen specific T cell proliferation ? how expensive is BrDu assay?

I want to clarify, whether you want to see the ag specific proliferation or t cell functional specific activation? If functional specific, ELISPOT is a good choice. lalitha kabilan

@ lakshimi: I think it was around 500-600 euros for 50 stainings (up to a few million cells per staining). Take a look at the eBioscience kits, they worked really well in our setups.

@ Lalitha, indeed ELISPOT cytokine analysis is good for functionality (IFN-gamma, IL-4, TNF-alpha etc) however, proliferation is also a sign of activation...

Ki 67staining kit for proliferation is a good assay but unfortunately it is also FACS based - it does however provide clear delineation of the cell population/s that are proliferating if that is important to your experimental results.