Plate method rather than flow cytometry. I'm mapping epitopes rather than following cell types.
Flow ICS is the best way as it is more quantitative.
MTT is OK. http://www.ebiotrade.com/buyf/productsf/promega/tb112.pdf
If the goal is epitope mapping rather than generating data, for CD8+ T cells, try to observe the T cells killing each other: you dope the peptides to be screened in the wells that contain your T cells. After overnight coculture, your T cells in the wells that contain the right epitope-containing peptides (assuming you are using overlapping longer than minimal length peptides to screen epitopes) will all go apoptosis (assuming your T cell purity is not too bad) which can be easily identified by eyes.
CFSE provides clear information about T-cell proliferation, it is also cheap and easy to use on T-cells
Thanks for your answers! CFSE is used with Flow, but I'm limited by my animal model--rabbit. There aren't as many reagents for those cells.
Dear Barbara, you can detect T cell proliferation by direct counting of blast cells. If you have access to flow cytometer, you can determine the percent of blast cells on FSC-SSC plots. In any case, you will not be confident that proliferating cells are T lymphocytes. So, if you have any possibility, change model and use inbred mouse strains! There will be fewer problems in the future.
Have you considered AlamarBlue? It's similar to the CellTiter product Weisan Chen recommends, and ought to work better than MTT.
http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/alamarblue-rapid-and-accurate-cell-health-indicator.html
Hi Barbara, I agree with Luke, Cell Titer or WST-1 are very easy to use and you can use them in 96-well plates (only need an absorbance plate reader). Both of them give you the chance to follow your cultures through time with no need for washing or extra plates (you can take your plate back to the incubator after reading it).
Dear Esmeralda and Luke, I should note that Alamar blue assay allows to estimate cell proliferation indirectly. This is redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity of cultured cells. MTT detects proliferation indirectly also, though.
Good point, Dmitry, it's important to build in a method for testing alternative interpretations into any experiment measuring proliferation. None of these technologies account for survival, and with any metabolism-based assay the output doesn't directly distinguish between activation and division. It's tedious, but counting the cells ought to catch any problems.
Thank you, Luke! Also, I don't see any obstacles to use CFSE in rabbit model, for exception restricted choice of additional mabs for flow cytometry.
Thanks Luke, Dmitry, Esmeralda and all! I am stuck with my rabbit model, and have looked at getting CyQUANT from Invitrogen or the other Promega and Agilent methods. I think I can use internal controls (no Antigen and ConA) to adjust for the indirect measurement methods. Luke, either activation or division would be OK for me since I want to know Yes vs No activation.
I'm just not finding a lot of references in this millennium. Everybody does CFSE with Flow cytometry. Have any of you used the dye/stain methods personally?
You can see description of the method and results on Fig 4B here:
https://www.researchgate.net/publication/233960011_Direct_Recognition_of_MHC_Class_I_Molecules_in_Memory_Response_to_Allogeneic_Tumor_Cells?ev=prf_pub
Article Direct Recognition of MHC Class I Molecules in Memory Respon...
CFSE, efluor 670, efluor 450 (or similar), if you have Flow Cytometer. Brdu assay if you want to use ELISA
I agree with others, you can use CFSE however do not forget use dead cell marker to exclud them from low fluorescence region
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