Re: Rodent thymocyte suspension

Dennis_Young@CIS.ucsd.edu
Wed, 9 Aug 1995 13:25:00 -0700

RPMI has a lot of calcium. You may be activating calcium-dependent
integrins, especially at high cell concentrations.
Try EDTA 1.0 nM in PBS without Ca++/Mg++.

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* Dennis J. Young Voice : (619) 822-0407 *
* Flow Cytometry Core Facility FAX : (619) 822-0412 *
* University of California, San Diego USA e-mail: djyoung@ucsd.edu *
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>>>>>

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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>From: Ralph Smialowicz 541-5776 <SMIALOWICZ@am.herl.epa.gov>
>Subject: Rodent thymocyte suspension
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