Hi Rupert,
How do these cells fare if they're put through the same process (including
sitting around for the same length of time) without going through the sorter
- would they be dying anyway?
It's generally best to keep the cells happy by sorting them suspended in
growth medium with whatever supplements they require - although this may
increase their tendency to clump. I haven't found fluorescence from media
such as RPMI to be much of a problem, and it's generally less inconvenient
than cell death. Remember to collect them into a nutrient medium as well,
perhaps enriched to allow for dilution with sheath fluid. If the sheath
fluid is a problem, try using an indicator-free medium (background
fluorescence from the sheath is more of a problem than fluorescence from the
medium used to suspend the cells).
You could also try dropping the laser power and perhaps the sheath pressure
and if the cells need to adhere to thrive, you could try sorting onto
coverslips rather than into tubes,
Ray
Ray Hicks
________________________________________________________________________
|University of Cambridge |Tel 01223 330149 |
|Department of Medicine |Fax 01223 336846 |
|Level 5, Addenbrookes Hospital |e-mail <rh208@cus.cam.ac.uk> |
|Hills Road Cambridge |Web http://facsmac.med.cam.ac.uk |
|CB2 | http://www.med.cam.ac.uk |
|UK |ftp server ftp://131.111.80.78 |
|_________________________________|_____________________________________|
> From: Gilbert Pitts <grpitt2@pop.uky.edu>
> Date: Fri, 23 Mar 2001 09:10:47 -0500
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Subject: Viability of FACS sorted cells
>
>
> Hi Everyone,
>
> I have recently tried to sort GFP-identified cells from the mouse
> suprachiasmatic nucleus. Unfortunately, the cells appear to be dead
> after the FACS sort is completed. I've been using a trypsin dispersion
> is HBSS (supplemented with Hepes and pen/strep). Does anyone have any
> ideas of how I can increase cell viability? Thanks in advance!
>
> Gilbert
>
> --
> Gilbert Pitts, Ph.D.
> Post-doctoral Scholar
> Dept. of Physiology
> University of Kentucky
> 800 Rose St, Room MS508
> Lexington, KY 40536-0084
> Tel. (859) 257-1645
> Fax: (859) 323-1070
>
>
>
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:12 EST