Dear guys:
I will start the CFSE Flow cytometry for Treg proliferation in next month. However, I am totally unfamiliar with the related parameters and cells number. Could you please recommend some protocols for me?
Thanks a lot.
Hi Shuoyang Liu
Take a look at these two protocols. They are very good and well explained. Good luck!!!
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185625/
http://www.nature.com/nprot/journal/v2/n9/full/nprot.2007.296.html
Hi Shuoyang Liu
Take a look at these two protocols. They are very good and well explained. Good luck!!!
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185625/
http://www.nature.com/nprot/journal/v2/n9/full/nprot.2007.296.html
Afternoon Shuoyang Liu,
Andre has identified two good papers. But a note of caution - we have found that excessive CFSE concentrations will label your cells and they will stay intact(alive) for 5-7 days but are unable to proliferate even to strong mitogens eg PHA.
Hello Shuoyang,
As you can see, the protocol is relatively simple. CFSE is toxic to cells. I would use couple of concentration, incubate at 37 for about 5 min, and inactivate CFSE by FBS.
Also, label cells with some general T cell marker or live stain before flow. You can use it to clean up the signal.
Actually, CFSE is not the best out succinimidyl esters. It bleeds a lot to other channels.
Good luck
Hi Shuoyang Liu,
We found that CFSE worked well when titrated, but users we have generally moved to CMFDA(488nm excitation), Cell Proliferation Dye eFluor® 670(633nm ex.) or eFluor® 450(407nm ex.), though you would need different excitation lasers to use each. These all give good retention and post-labelling viability, titration is still required, allowing more staining parameter flexibility, plus allows them to track two populations independently.
Best wishes
Andre's references are great. I agree with Martin and Denis' warnings of CFSE toxicity. I would add that a higher cell density during CFSE staining and higher cell density or U-/V- bottom plates (to increase cell-cell contact) when seeded for culture afterwards, will increase chances of cell survival. Best of luck.
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