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6 Total and viability count
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6 Total and viability count
The NucleoCounter detects fluorescent signals from propidium iodide (PI) when it is
bound to double-stranded DNA (dsDNA). The fluorescent intensity of PI is increased
substantially upon binding to dsDNA. As a result the NucleoCounter detection is
highly specific and sensitive. The staining of the dsDNA requires that PI can
penetrate the cell membrane. As with several dyes used for the staining of cells the
cell membrane of living cells is impermeable to PI while dead or damaged cells are
readily stained with PI.
6.1 Non-viable Cell Concentration
In analogy with several other dye exclusion methods, such as methylene blue
staining, the NucleoCounter method uses dye permeability for the determination of
non-viable cells. Thus by measuring sus pended cells under conditions where
viability of the cells is retained, only cells directly stained with PI are assumed to be
non-viable. Therefore when determining the concentration of non -viable cells the
cells are diluted by an isotonic solution, or any other solution that preserves viability
of the cells, and then the NucleoCassette is loaded with this solution. Since PI does
not stain viable cells only non-viable cells are detected, resulting in the
determination of non-viable cell concentration.
6.2 Total Cell Concentration
In order to determine the total cell concentration it is necessary to subject the cells
to a treatment that renders the cell membrane permeable to PI. This is most easily
done by diluting the cell suspension with Reagent Y100, which is an effective cell
lysing agent. Other methods of lysing the cells can also be used, which do not affect
the staining and/or fluorescence efficiency of PI. For recommended method for
lysing cells please refer to appropriat e application note.