ChemoMetec YC-100 - Manual (Page 39)

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 suspended 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 appropriate application note.