Dear Bahareh,

please see my attached protocol for CFSE staining. You can definitely titrate the CFSE down to 1 μM and still get a good signal. It's a rather exhaustive step by step protocol, which also provides with a good method for quenching. I have used it for all kinds of mouse cells, but am pretty confident that it works for human cells, too. Please check it out!

All the best and good luck with your project,

Michael

Dear Michael and Zaid,

Thanks a lot foryour responses and your protocols. I will run FACS tomorrow and  then I can inform you from my results. 

Best regards

Bahareh

Dear Michael,

I have another question,  should I wash my cells before running FACS by centrifuging method or Can I run FACS direct? your protocol includes washing step,  by this step is CFSE stayed attached to cells and  what happen if I dont wash cells? thanks again for your help.

Hi Bahareh,

CFSE is cell permeable and couples to intracellular proteins via its succinimidyl ester group. The additional washing step before acquisition is just to remove excess CFSE. You absolutely need not to worry that you will wash it off or loose signal, as a) it's in the cells and b) gives such a bright signal in the FITC channel/ green laser, it's rather "too much of a good thing" and requires careful compensation. Hope that helps!

All the best and good luck for your experiment!

Michael

Hi Michael,

Thanks for your response I tested with centrifugeing and it worked. Thanks alot for your help.

Good luck with your project

best regards

//Bahareh