Re: Rodent thymocyte suspension
Rochelle A. Diamond (diamond@cco.caltech.edu)
Wed, 9 Aug 1995 12:29:43 -0700 (PDT)
Ralph- I have been working with mouse thymocytes for over 12 years now.
They seem to respond badly to PBS. Try switching to Hank's Balanced Salt
Solution with 2.5mg/ml BSA, 20-100ug/ml DNAase and 5mM azide. When we
count thymocytes in PBS with and without BSA as a matter of fact they take
up eosin because they become leaky. In HBSS w/BSA they do not become
leaky and do not die in mass. We make monodispersed suspension by
harvesting the thymus in RPMI 1640 with 2.5mg/ml BSA 2mM Glutamine and
10mM Hepes pH7.0 then pushing them thru stainless steel wire mesh fabric
from Tetco in NY in a petri (not TC dish) keeping them wet with media. We
transfer from the petri with quantitative rinse then spin them at 200g for
10min at 4C and resuspend in the same media or Hanks as above with a 5 ml
serological pipet. I get no clumping even at 100 million/ml for
elutriation. If you do see a clump as I sometimes see with spleen T
cells, I resuspend and let the tube sit for 15-20 sec, then gently pour
out of the tube into a fresh tube, leaving the clump(s) behind. I rarely
need to do this however.
Let me know if I can be of further assistance.
Rochelle Diamond
California Institute of Technology
Division of Biology
Flow Cytometry/Cell Sorting Facility
diamond@cco.caltech.edu
On Wed, 9 Aug 1995, Ralph Smialowicz 541-5776 wrote:
> 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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