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

Paul M. Donohoe p-donoho at nimr.mrc.ac.uk
Thu Aug 25 10:24:03 EST 1994


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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