Cell Counting: Viability

tylee tylee at ITIS.COM
Mon Sep 29 22:57:50 EST 1997


Karyn,
Like you, to determine cell number over the years (doing mostly
proliferation assays), I have counted hundreds of samples using a
hemocytometer (leading to microscope headaches), followed by thousands of
samples using a Coulter Counter (also tedious), then followed by tens of
thousands of samples/column fractions relying on incorporation of tritiated
thymidine (labor intensive to harvest, radioactivity hassels, etc.).
Other assays I have tried but would not recommend include methylene blue,
neutral red, sulforhodamine B all requiring washing the cells before
recording data, which is just about as labor intensive and prone to
increases in variability among replicates as the thymidine incorporation
assay.
My recommendation for an easy approach to obtain an estimate of the number
of viable cells in large numbers of samples is to use a tetrazolium assay.
Viable cells convert tetrazolium dyes to a colored formazan product that
can be read with a spectrophotometer (or a 96 well plate reader for larger
number of samples). The MTS tetrazolium compound is the easiest one to use
because it is converted to a aqueous soluble formazan product that can be
recorded directly in the same medium used to culture the cells. You just
add the reagents available in a kit, incubate, and record absorbance at
490nm. You can do the tetrazolium assay using cells cultured in a 6 well
plate, then transfer to a cuvette and read; but, it is faster to grow the
cells and do the tetrazolium assay and record data all from the same 96
well plate. Under most experimental conditions, the absorbance is directly
proportional to the number of viable cells; however, if you are comparing
cells at near clonal density in log phase growth to those that are piled on
top of each other (as you mentioned), I would recommend running a standard
curve to ensure that the metabolism of the cells (ability to reduce the
tetrazolium compound) is not drastically different between the 2
populations. 
My two cents worth.
Ty Lee



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