I have some suggestions that perhaps you can try:
1) use ACK lysing buffer.....I used it for 15 years for mice (pbl, spleen
and bone marrow), and now use it for monkey blood....easy to make at 10x
which keeps for 4 months (or more) and is made up to 1x in H20 and used at
room temp.
2) There is a mouse ficoll-based product called "Lympholyte-M" specifically
meant to be used with mouse blood. It is distributed by Cedarlane/Accurate
chemical.
3) Do a blocking step of the FC receptors by blocking with "FC-block", a
CD16-32 (?), 2.4G2 clone from Pharmingen meant to reduce this sort of
autofluoresence in mice.
Tricia
*****************************************************************
Patricia L. Echeagaray
Flow Cytometry Services
Serquest.....a Southern Research Institute Company
431 Aviation Way
Frederick, MD 21701
USA
Voice: 301-694-3232 ext 276
FAX: 301-694-7223
echeagaray@serquest.com
*****************************************************************
> -----Original Message-----
> From: Joern E. Schmitz [SMTP:jschmitz@caregroup.harvard.edu]
> Sent: Friday, July 09, 1999 5:31 PM
> To: Cytometry Mailing List
> Subject: Lysis of erythrocytes / streptavidin conjugation with "low
> backgrounds"
>
>
> Hi Everybody,
>
> Recently we are including mice as another animal species into our
> studies. Lysis protocols for whole blood specimens or lysis of
> erythrocytes in spleen fractions however seem to be much more cumbersome
> than in specimens from human or monkeys. Seperation of cells by
> gradients (ficoll) seem to be different than seen in human or monkey
> specimens.
>
> Eventually, we want to perform "tetramer-staining" on mice specimens.
> However, the streptavidin conjugates which we tested so far have quite
> some background staining (between 0.2 and 2%) on mice T cells (CD4+ T
> cells have even a higher background than CD8+ T cells.)
>
> Does anybody out there have some good suggestions for:
> 1) Lysis of mice erythrocytes
> 2) gradients like ficoll specific for mice T lymphocytes
> 3) streptavidin conjugations to PE that have a "very" low background
> staining on mice T lymphocytes
>
>
> Thanks for your help. Depending on the amount of responses I will put
> out a summary on the web.
>
> Joern E. Schmitz, M.D.
> Division of Viral Pathogenesis
> Beth Israel Deaconess Medical School
> Harvard Medical School
> Boston
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