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FACS kills my CFUs!
Posted by: pombetom (IP Hidden, New member, 3)
Date: September 23, 2005 10:39AM

Hi friends,

I would greatly appreciate any insights you might have for this problem:

When trying to culture cells from bone marrow aspirates that have been sorted by FACS for various cell surface markers, the ACT OF BEING SORTED by the FACS machine leads to NO growth of colonies. I've performed control experiments for every step of the protocol, including a sample of cells loaded into the "hopper" of the FACS machine immediately prior to sorting. I get colonies from that sample, but NOT from cells that have passed through the machine. Granted, the fraction of CFUs in this population is quite small, but I can routinely get 15-50 colonies from non-sorted samples, and ALWAYS zero from sorted samples. It makes no difference if the cells are sorted into media or 100% serum. My last remaining hope is to use a FACS nozzle of a larger diameter.

Has anyone heard of such a problem, and do they know of a solution?

Many thanks.

 

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Re: FACS kills my CFUs!
Posted by: jonmoulton (IP Hidden, Junior member, 15)
Date: September 23, 2005 01:38PM

Do the cells die of you send them through with the laser turned off? That would at least control for photochemical effects.

- Jon

 

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Re: FACS kills my CFUs!
Posted by: pombetom (IP Hidden, New member, 3)
Date: September 23, 2005 02:13PM

Thanks for the question. I agree that such a test would be useful, if the machine allows. Of course, I won't be too happy if that is the source of the problem, because it's my understanding that the laser is rather important for the sorting part...! It might help if the intensity of the beam can be turned down.

Cheers.

 

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Re: FACS kills my CFUs!
Posted by: ramanaedu (IP Hidden, New member, 1)
Date: April 11, 2006 06:24AM

The presence of azide in the sheath fluid might have prevented the colony formation. Try using sterile PBS alone as sheath fluid.

 

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Re: FACS kills my CFUs!
Posted by: pombetom (IP Hidden, New member, 3)
Date: April 11, 2006 09:26AM

Thanks for the suggestion, Ramanaedu, but there is no azide in the sheath fluid.

 

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