Hi all,
I was wondering whether someone has already experienced a single cell isolation (cloning) with HEK293 cells which have been stained beforehand with CFSE or CMFDA ? The FACS is a FACS Aria 3.
Will the cells support the sorting once they have been stained with the tracker, let's say the day before ?
Many thanks !
Alexis
I know that CFSE-stained T cells keep dividing in short-term cultures. On the other side CFSE and CMFDA should modify all free amino groups and negatively affect cell survival and proliferation. That means that success of single cell cloning of CFSE-labeled cells will depend on the extent of modification (brighter cells equals less cells to survive) and number of cells. The more cells you will start with - the higher is the chance to get clones.
Hi Yuri, and thanks for your help.
You're right, I'm afraid that the slight toxicity of CFSE might impact the survival rate of cells when associated to the stress of the sorting. CMFDA is a bit less toxic, so it might improve slightly the recovery...
We have used CFDA to stain bone marrow derived macrophages and injected these stained cells to peritoneal cavity of mice. Next day, we recovered them from peritoneal cavity and did FACS.
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