Re: FSC/SSC interpretation

Stuart:
7AAD is a membrane impermeable DNA dye. As such there are things it can show and others it cannot, and that's the important thing to realize, in my opinion - what is what your test can tell and what it cannot tell. It can certainly be blind to cell death processes that occur earlier to membrane integrity problems. On the other side, it is a very good stain for what it does. What is viability, what is the metabolic state you want your cells to be? That is up to you to decide beased on your demands. You could try to use PI as well. Since PI is brighter it will give you more "resolution" to look at your cells' uptake of the dye. In our experience and according to our findings, cells can show PI fluorescence in a range of intensities and not just positive negative, and similarly to 7AAD the dimmer cells are earlier in the apoptotic process. 

If you want to deepen your understanding of the death biology of your cells (since it seems that is important to you), you can try other markers as well. AnnexinV is a a good example. The problem is that you need a centrifugation step and if your cells are very fragile or in a "dynamic death process phase" that could be a source of bias. In our experience, the most direct way to look at the cells is with a mitochondrial membrane stain such as DIOC or TMRM. We add the dyes directly to the culture well, incubate in the dark and then take the cells, add PI (or 7AAD for TMRM), wait 10 min and immediately acquire, without centrifugation and without delays. That is as close as we can get to "seeing" the actual state of the cultured cells, and it works very well (DIOC with PI is a favorite). Take care to titrate the concentration of your mitochondrial dyes, which should work at the low NANO-molar range. If you use too much they'll start to depend on membrane potential, which although can also be used as a measure of apoptosis, it is not only for apoptosis and besides, you will not be looking at what you think you are looking. There are many good references on the subject; H. shapiro's book was an excelent start for me, and I always liked this article specially for it's treatment of the "JC-1 controversy" (Eur. J. biochem. 264:687, 1999).

Regards,

Uriel.

Uriel Trahtemberg
MD/PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL 
 

"Put your hand in a hot stove for a minute, and it seems like an hour. Sit with a pretty girl for an hour, and it seems like a minute. That's relativity."
 Albert Einstein.
----- Original Message ----- 
From: Stuart Berzins 
To: cyto-inbox
Sent: Thursday, July 28, 2005 5:21 AM
Subject: FSC/SSC interpretation


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 
supplements.

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
-- 
Stuart Berzins Ph.D,
Research Fellow, Godfrey Laboratory,
Department of Microbiology and Immunology,
The University of Melbourne,
Parkville 3010,
AUSTRALIA.
email: berzins@unimelb.edu.au
Ph: +61-3-8344-5704
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