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I am analyzing B lineage cells in multiple myeloma patients for increases in
DNA content using crbc as an internal control and with multi-color
immmunofluorescence am staining both T and B cells in the same sample. We have
arbitrarily decided to call T cells diploid and characterize a B cell as
aneuploid only if it has a DNA content greater than that of the T cells. DNA
is stained with DAPI. We get cv's in the range of 2-4 for diploid cells.
My question is as follows: How can one distinguish whether a population of
cells at approximately the 4N channel range is cycling in G2/M, or whether it
is arrested in a tetraploid state and represents a 4N DNA stemline? We will be
sorting these cells and looking at them in cytospins to assess mitotic cells,
but is there a less laborious way to check this for every sample analyzed? We
do about 5-10 patients each week. Is the proportionate relationship between S
phase region and the G2/M region a decent indicator? For thymocytes, the % S
phase cells is approximately equal to the % G2/M. For many patients with a
large near-4N peak, there are few S phase cells.
I would appreciate any advice on this. It might be of interest to others that
four color staining (FITC, PE, Tandem, and Dapi) works beautifully and allows
precise analysis of the phenotype of lymphocyte subsets.
Thanks,
Linda Pilarski, Department of Immunology, University of Alberta Edmonton T6G2H7
Canada FAX (403) 492-0368 Lpilarsk@vm.ucs.ualberta.ca
Regards,
Linda
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