Absolute NK cell count in SCID mice
petter.hoglund at ki.se
Sat Mar 24 19:38:24 EDT 2007
Dear Susann, CD56 is not expressed in mice. I would use an antibody against
NKp46, which is the most specific NK cell marker know so far. It is
expressed i all strains tested so far. R&D systems has a polyclonal goat
antibody that works well in FACS (their rat monoclonal does not seem to work
as well). Alternatively, you could use an antibody against IL-2Rb, which
also stains NK cells from all strains, but is slightly less NK-specific. We
use both these antibodies in combination with CD3 in normal mice. You may
not need CD3 that in the SCIDs, unless they are a little older and contain
some T cells. Blood is fine for NK cell identification. In normal mice you
find 1-5 %, in SCIDs the percentage should be higher. Good luck /Petter
Department of Microbiology Tumor and Cell Biology (MTC)
S-171 77 Stockholm
Tel: +46 8 52 48 62 01
Fax: +46 8 30 42 76
E-mail: petter.hoglund at ki.se
> -----Original Message-----
> From: Wilson Mandala [mailto:wmandala2002 at yahoo.co.uk]
> Sent: den 23 mars 2007 09:26
> To: Cytometry Mailing List
> Subject: Re: Absolute NK cell count in SCID mice
> Dear Susann,
> I dont have much experience with blood samples from SCID
> mice, but working with human peripheral blood we have used
> CD56-FITC, CD16-FITC, CD3-PerCP and CD14-APC for identifying
> NK cells.
> The CD56+CD16+CD3- are then described as proper NK celss,
> with CD56+CD16+CD3+ cells as your NKT cells and the
> CD56-CD16-CD3+ as your T cells. The CD14-APC just makes sure
> that you eliminate any monocytes from your lymphocyte population.
> Basically the procedure is to stain peripheral whole blood
> (25ul) with 2ul of each monoclonal antibody (we have used BD
> antibodies). Incubate at 20oC for 15 minutes. Lyse (2ml of 1x
> FACS Lysing solution) for 10 minutes. Spin at 1500 rpm for 5
> mins, aspirate the supernatant, wash with 2ml of PBS and
> centrifuge at the same speed. Aspirate supernatantand fix
> cells in 1% Formaldehyde + PBS solution and acquire. We have
> had good results with such method.
> Good luck.
> Wilson Mandala.
> Research Scientist,
> Wellcome Trust,
> Blantyre, Malawi
> --- "Szmania, Susann M" <SzmaniaSusannM at uams.edu>
> > We want to determine the absolute number of NK cells in the
> > blood of SCID mice. Does anyone have a procedure?
> > Antibody recommendation? The
> > strains we want to test are:
> > C.B-17/IcrHsd-Prkdc<scid> and
> > IcrTac:ICR-Prkdc<scid>
> > We appreciate any input.
> > Thanks,
> > Susann Szmania
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