Dear Ms. Rezalotfi,

I am not experienced in cancer research, but to analyze the different functional aspects of T cells, multi-color flow cytometry may be a standard. Generally speaking, we stimulate T cells with peptides or by coculturing with cells bearing the target antigen for around 6 hours. After the stimulation, cells are collected and surface/intracellularly stained for flow cytometric analysis. Related methods are described in a number of publications including the following.

https://www.nature.com/articles/nprot.2006.268

Regards,

Hi Alaleh,

That would depend on your model, do you use murine or human T cells? In addition to what Tsukamoto said, traditionally, the gold standard for analyzing effector T cell cytotoxicity is the radioactive chromium (51Cr)-release assay. However, its major disadvantage is the use of radioactivity and for some labs this is not practical. Alternatively, you can use tracking dyes such as CFSE and evaluating viability if your lab has access to a flow cytometry (DOI 10.1007/978-1-61737-950-5_7). Another possibility is analyzing T cell degranulation using CD107a (Chapter CD107a Degranulation Assay to Evaluate Immune Cell Antitumor...

Dear Alaleh Rezalotfi

I just briefly wanted to email your concerning your question. If you wanna avoid radioactivity (chromium (51Cr)-release assay), maybe the following paper helps as it provides a protocol for a FACS based assay based on CFSE dilution and 7-AAD viability stains.

Article Development and Application of a Multiplexable Flow Cytometr...

All the best and good luck with your experiment,

Michael

Thank you all for sharing your helpful experiences. I have done multi-color flow cytometry for the favorable T cell markers (for human) in addition to T cell expansion through CFSE labeling and now need an additional functional assay to confirm the previous ones. I haven't heard about radioactive chromium (51Cr)-release assay for assessing cytotoxicity of T cells (and NK cells), whereas I am going to perform lactate dehydrogenase (LDH) releasing assay for the target cells. That was so interesting, however it would not be practical in our cellular lab.

Heriberto Prado-Garcia Thank you for recommending CD107 marker and that interesting chapter. That would be very helpful.

Best,

Alaleh