INF g and TNF b intracellular staining

From: Michal Abel (abelm@necker.fr)
Date: Fri Apr 12 2002 - 14:36:10 EST


Dear experts in flow cytometry,

I would like to perform INF g and TNF b intracellular staining of a
small (0.1%) tetramer + PBMC.
I have some doubts and I would be grateful if you could give me any suggestion.

My lymphocytes are frozen and I wonder if there are any particular
conditions for its stimulation with specific peptide.

The other thing that bothers me is the precision and specificity of
facs analysis. My population of Tetramere + cells is already small
and its subpopulation of INF g + will be even more difficult to
distinguish from the background staining. I wonder if it would be
possible (and correct) to culture the lymphocytes after peptide
stimulation during 48h to increase the number of double positive
cells without changing considerably the proportion of tetramer+/ INF
g + cells?

Thanks for any suggestions!

Michal
--
Michal Abel, MD, PhD
Laboratory of Clinical Immunology
INSERM U25
Necker Hospital, 161 Rue de Sèvres. Paris
tel: 33 1 40 19 28 87
fax: 33 1 44 49 53 74



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