HELP! Why are my T cells normal by FACS?????

mdcabl at cc.newcastle.edu.au mdcabl at cc.newcastle.edu.au
Fri Aug 26 06:55:50 EST 1994


Hi,
Just wondering. Why do you think your results are incorrect?
Allen Black
Dept. of Pathology
Univ. of Newcastle

In article <33id2j$drg at mercury.hgmp.mrc.ac.uk>, p-donoho at nimr.mrc.ac.uk (Paul M. Donohoe) writes:
> I am examining the changes in immune system parameters over a time
> course in rats with Adjuvant Arthritis, and in r65kD-protected rats,
> one part of which is looking at lymphocyte surface markers by
> immunostaining and FACS.   All the antibodies I've used are commercial,
> and all give nice, discrete populations, with the correct levels of
> expression.   I have used MAbs against CD2, CD3, CD4, CD5, CD8, CD25
> (IL2R), CD44, CD45RC, ab-TCR and RT1B (MHC class II).
> However, during the disease when there is a high level of inflammation,
> and in the protected groups, also presumably immunologically active,
> there is NO DIFFERENCE AT ALL in the levels of any of these markers,
> either by percentage of population, or mean intensity of fluorescence,
> from those of normal rats.   That is, differences of about 1%, less
> than standard errors of 1.5%.   The arthritic rats showed normal
> disease progression, and the protected rats normal protection.   After
> sacrifice, the lymph nodes and spleen were removed, placed in cold PBS,
> and transported to me on ice, a delay of about 2 1/2 hours.   I
> immediately washed, counted and stained the cells, and fixed them for
> FACSing the next day.
> Has anyone else experienced a similar problem, or have any advice to
> give me?   From the couple of papers I found concerning temporary
> storage of lymphocytes, a few hours on ice should not have made a
> difference.   Also, although not the actual site of disease, the
> draining lymph nodes [popliteal (knee) and inguinal (flank)] were
> swollen, indicating influx of (presumably) lymphocytes and macrophages,
> at least.   I've torn all my hair out, and scratched my head raw trying
> to fathom it.....HELP!
> 
> Please reply by mailing me at p-donoho at uk.ac.mrc.nimr or by posting a
> reply here at bionet.immunology.   Many thanks in advance for any help!
> 
> Paul M. Donohoe
> Leprosy Dept., NIMR, The Ridgeway, Mill Hill, London NW7 1AA.
> PhD Student



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