Mouse Spleenocytes

Mike O'Brien mikeobrien at btinternet.com
Thu Mar 21 15:07:44 EST 2002


Dear Flowers,


I have been working with Flow cytometry for the last 3 years but have only
recently become aware of this discussion group.  I am investigating the
effects of increasing tumour burden on mouse spleenocyte subsets and
function.  On initial flow analysis there are two main populations of cells
1) the morphologically lymphocyte population and 2) a second population that
have a similar side scatter profile but a smaller forward scatter profile.
This suspicious population of cells  constitute 25 - 40% of the total
population.  However overall the cell viability by Trypan Dye Exclusion was
95%+.  When non-fixed cells were analysed for permeability to Propidium
iodide 16% of the suspicious population took up PI as compared to 3.5% of
the morphologically lymphocytic population.  When examined for
phosphatidylserine expression with Annexin V 5% of the suspicious population
were positive as compared with 1% of the lymphocyte population.  When
analysed for T cell antigen expression 57% of the lymphocyte population
expressed CD3 as compared to 9% of the suspicious population.  My question
is have other investigators found this population of cells also?  From the
published reports I have not seen any reference to them.  Would the
consensus opinion be that these are dying rather than dead cells?  I have
looked at every aspect of cell preparation and they are a constant finding.
Could they represent immature cells from the splenic follicles?  I would
apreciate any advice as to how to move this forward or if previous workers
have encountered the same problem what there conclusions were.

Thanks for your help.

Mike O'Brien
Dept. Of Microbiology,
University College Cork,
Cork,
Ireland.




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