FSC/SSC interpretation

Stuart Berzins berzins at unimelb.edu.au
Wed Jul 27 22:21:44 EST 2005

Hello. We are studying the function of some immunoregulatory mouse T 
cell subsets by culturing splenocytes with (or without) a variety of 
different stimuli for 3 days. These cells can  produce high levels of 
cytokines and divide extensively. It is extremely important for us to 
know whether the cells at the end of the culture are 'healthy' and 
therefore, likely to have functioned normally throughout the culture 
period, so that we can interpret the effects of various culture 

Our main viability test is 7-aad  (~4ug/ml) and we only include the 
'most-negative' population for analysis. The 7aad-intermediate 
population thought to correspond to apoptotic cells is also excluded. 
How confident can we be that the 7-aad -low cells are viable and 
healthy? We ask this question because there is often a high 
non-viability rate in the cultures. Our primary concern is that our 
culture conditions may not be optimal and that our 'viable' cells may 
just be 'slow' to die and don't reflect normal function.  We are 
concerned because the cultured cells usually look a bit unwell at the 
end of the culture (although given the low viability rate, this is 
not entirely unexpected), and because the FSC/SSC  profile of the 
viable cells often show higher than normal SSC ( FSC looks normal).

Conversely, some co-workers argue that the 7aad-negative 'viable' 
cells are, by definition, viable and that given their 7aad-negative 
status we can safely assume that they have been healthy for the bulk 
of the culture time, even if hey are starting to look a little 
unusual by FSC/SSC at the end of the culture (high ssc). Is it safe 
to trust 7-aad as a gold standard for viability and, or should we 
incorporate additional tests?

Stuart Berzins Ph.D,
Research Fellow, Godfrey Laboratory,
Department of Microbiology and Immunology,
The University of Melbourne,
Parkville 3010,
email: berzins at unimelb.edu.au
Ph:	+61-3-8344-5704
Fax:   +61-3-9347-1540
Mobile: 0427 849 123

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