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Extracting DNA from FACS sorted cells - (Sep/20/2011 )

Hi,

I am sorting a rare population of cells by cytometry... I am getting the sorted cells in a volume of 5 ml... I have tried this a couple of times to make a cellular pellet from this 5 ml... Unfortunately, I am loosing a lot of cells in the supernatant... Thus my yield of DNA is very small... Can anyone please tell me, how I could get the cell pellet without losing much cells after cytometry... I would be extracting DNA from this pellet...

I will appreciate any help in this regards...

Thanks,
Basudev

-Basudev Chowdhury-

What type of cells are they? And what are the centrifuge settings you used?

-leelee-

I don't really understand why your cells will remain in the supernatant, however you should be able to pellet them down by increasing the speed or the time (or both).

On the other hand, as you are going to lyse your cells to extract the DNA, I assume viability is not an issue, so you could always use harsher ways. You can centrifuge them in a benchtop micro-centrifuge. Either split the 5ml in 5 x1.5ml tubes, or spin 1ml at a time. 5min at max speed should definitely pellet the cells. Remove the supernatant, and resuspend in lysis buffer. I do that to prepare lysates for protein analysis and so far has never failed me.

-almost a doctor-

The solution to the problem was already put forth by "almost a doctor." split the collected volume and spin. The reason this happens has been a point of conjecture for years. The best explanation I've heard of goes as follows. All the droplets containing cells that are deposited in your collection tube share the same charge. This build-up of charge causes the cells to be held up in solution and not pellet. One way people have tried to get around this is to transfer the liquid to another tube and therefore dissipate the charge. You could also insert a copper wire and connect it to something metal to act as a ground and allow the charge to flow out of the suspension.

-RynDggn-