Cell viability can also be determined by the vital dye exclusion assay. Dead cells have lost plasma membrane integrity and as a result cannot exclude dyes and thus are stained. For example, trypan blue can be used to stain yeast. The ability of cells to exclude trypan blue indicates that they are still alive. Thus, if yeast failed to grow on galactose plates but still excludes trypan blue, then presumably this is the result of a problem with the cell cycle rather than cell death. On the other hand, inability to exclude trypan blue (cells are thus stained blue) indicates that the gene introduced is lethal to yeast. The percentage of blue cells can also be employed as an indicator of the potency of the killing. Other vital dyes, such as methylene blue and erythrosine B, have also been employed (62).
1. Grow YEp51-Bax-containing yeast and transfer to galactose-containing medium as described above for the clonogenic assay.
2. At different times after inducing Bax expression in galactose-containing liquid medium, remove 20 |jl1 of the yeast culture and mix with 20 jjuI of 0.4 % of trypan blue solution in PBS (Sigma). The numbers of dead (blue) and live (no colour) cells are determined using a haemocytometer (Hausser
Scientific, Horsham, PA) under a light microscope and the percentage of dead cells is calculated. The existence of cell wall on yeast does not interfere with trypan blue assays.
When the death gene introduced is under the control of a constitutive promoter, e.g. the ADH1 or GPD promoter, no transformants or far fewer transformants will form after introducing the expression plasmids into yeast and plating on selective drop-out medium, which selects for the introduced plasmid. An 'empty' vector should also be transformed in parallel as a control. In this assay, however, one will not be able to tell the difference between growth arrest and cell death. One way to circumvent this, is to introduce, for example, ADHl-Bax-bearing plasmid into a strain already containing an anti-apoptotic gene under the control of a conditional promoter, e.g. GAL-bcl-2. When cells are cultured in galactose-containing medium, expression of the anti-apoptotic gene will protect yeast cells from the cytotoxic effect of the introduced box gene. After transformants are formed on galactose plates, these can be streaked or patched on glucose-containing plates where expression of the anti-apoptotic bcl-2 gene is shut off, and thus the effect of the bax gene is revealed. In this case, streaking or patching in parallel to a glucose plate provides a control for any non-specific growth failures due to unforeseen technical problems.
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