Absolute NK cell count in SCID mice

Petter Höglund 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

Petter Höglund
Department of Microbiology Tumor and Cell Biology (MTC)
Karolinska Institute
Box 280
S-171 77 Stockholm
Sweden
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>
> wrote:
> 
> > We want to determine the absolute number of NK cells in the 
> peripheral 
> > 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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