Re: Rodent thymocyte suspension

RICHARD L. DARLEY (WHGRLD@cardiff.ac.uk)
Thu, 10 Aug 1995 16:36:38 GMT

These guys just love to apoptose - this might be your
problem. Keeping them on ice throughout prep, staining and analysis
might slow them down (i.e. azide might not be enough).
If you have a lot of clumping your concentration of DNase might not handle it -
try keeping the volume low (0.5-1ml) and up the concentration of enzyme until it
clears then dilute it out.

Good luck,

Richard Darley
UWCM
Cardiff

> Date: Wed, 9 Aug 1995 09:10:10 EST
> From: Ralph Smialowicz 541-5776 <SMIALOWICZ@am.herl.epa.gov>
> Subject: Rodent thymocyte suspension
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Priority: normal

> I would appreciate any help with a problem that we have had in preparing
> "clump free" thymocyte suspensions, from mice and rats, for flow
> analysis. We routinely prepare thymocyte suspensions in RPMI followed by
> centrifugation at 200 x g. Cell pellets are resuspended in PBS with 2% FBS,
> 0.1% sodium azide and 0.025% DNase, the latter is added to "prevent"
> clumping. The cells are subsequently centrifuged and resuspended in PBS
> with 2% FBS and 0.1% sodium azide for staining. If we have a clumping
> problem, it usually occurs after the first wash. In general, the clumping
> appears to be associated with cell suspensions with higher than usual cell
> numbers. Any suggestions would be greatly appreciated. Thanks.
>
> Ralph J. Smialowicz
> US EPA
> RTP,NC
>
>
>

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