I need to get clones from my nucelofected MEL1 cells (hESCs). However, I´d rather sort single cells in a 96 well plate by FACS than pick the colonies manually. Has anyone of you experience in this matter or could provide some substantial literature? Would the use of conditioned media enhance single cell survival? Any comments on survival and efficiency? I guess it will need quite an amount of cells to get a decent amount of growing clones.
Thank you very much for your help!
I have no experience in sorting hESCs, however, I would suggest to sort using a larger nozzle (100um) and low pressure (12-15 PSI) to ensure viability.
Yes, they will survive FACS. You will need to ensure there are enough cells when you plate them afterwards, so yes, you will need a lot of cells. To enhance survival, you can try adding Y-27632 (rock inhibitor) to the medium when plating (if you're not doing it already).
The alternative method is limited dilution to get single clone if the cells is very sensitive to physical stress. After dilution, check under fluorescence microscope to screen the positive clones. A bit tedious, but less chance to kill the cells or get contaminated.
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