Re: Viability

Mario Roederer wrote (in response to Stuart Berzins):
> Stuart,
> 
> in my opinion, PI (propidium iodide) is still the King of viability
> dyes...  7AAD is pretty good.  We use PI whenever possible.
> 
> However, these dyes don't work when you fix & permeabilize cells -- they
> leak out too quickly.  Thus, the fixable viability dyes (which are
> covalent reactions) need to be used... this includes EMA (the original),
> and the newer amine-reactive dyes from Molecular probes.
> 
> With regard to simple fixation (paraformaldehyde) -- we found as you did
> that PI is compatible with PF fixation ... for a limited time.  If   
> you analyze your cells within 2-4 hours, there is no significant
> problem.  But if you wait longer than that, the PI slowly leaks out of   
> the dead cells (and into the previously live cells) rendering the
> separation far less believable.
> 
 Actually, 7-AAD can be made to work well with fixation/permeabilization
if actinomycin D is added:

Schmid I, Ferbas J, Uittenbogaart CH, Giorgi JV. Flow cytometric
analysis of live cell proliferation and phenotype in populations
with low viability. Cytometry. 1999; 35:64-74.

BACKGROUND: Combined analysis of DNA content and immunofluorescence on
single cells by flow cytometry provides information on the proliferative
response of subpopulations to stimuli in mixed cell preparations;
however, in low-viability cell preparations, dead cells interfere with
accurate flow cytometric data analysis because of nonspecific binding of
antibodies and altered DNA-staining profiles. Light scatter differences
between nonviable and viable cells are unreliable, particularly after
the cell permeabilization step that is necessary for DNA staining. We
developed a method for identification of nonviable cells by
fluorescence in cell preparations that are stained simultaneously for
cell surface or intracellular immunofluorescence and DNA content.
MATERIALS AND METHODS: Nonviable cells that have lost membrane integrity
are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of
7-AAD from stained nonviable cells to unstained viable cells after
permeabilization is prevented by blocking DNA binding with
nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for
DNA staining because the orange spectral emission of PY can be separated
from the green fluorescein isothiocyanate (FITC) emission and the red
emission of 7-AAD, respectively.
RESULTS: Application of the method to the analysis of the T-cell
leukemia cell line Molt-4f and of cultured human peripheral blood
mononuclear cells is presented. In both cell preparations, 7-AAD
staining permitted reliable dead cell exclusion. Live, 7-AAD-negative
Molt-4f cells showed higher expression levels of cell surface CD4 and of
intracellular CD3, showed a higher proportion of cells in the G1 phase
of the cell cycle, and showed a lower coefficient of variation of the G1
peak compared with data obtained from all the cells in the preparation.
Live, CD8+ lymphocytes from OKT3-stimulated cultures of human
peripheral blood mononuclear cells showed a specific proliferative
response as measured by DNA content analysis.
CONCLUSIONS: The results show that cells stained with FITC-labeled
antibodies can be analyzed by single-laser flow cytometry for DNA
content combined with dead cell discrimination. Furthermore, they
emphasize the need for exclusion of dead cells from the analysis of cell
preparations with low viability to obtain reliable data on
immunofluorescence and cell-cycle distributions.

-Howard