Encyclopedia of Experiments: Biological Techniques
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Differential Nuclear Staining Assay - ExperimentTranscript
Mitocans are a class of drugs that selectively affect the functioning of mitochondria in cancer cells and induce apoptosis.
To determine mitocan cytotoxicity, seed a cell suspension containing semi-adherent cancer cells onto a multi-well plate. Treat the plate with the desired mitocan solution and incubate.
Mitocan enters the cancer cell to disrupt its mitochondrial activity, eventually leading to cell death in mitocan-sensitive cancer cells. Treat the cells with a staining solution containing a mixture of Hoechst dye and propidium iodide.
Hoechst, a membrane-permeable fluorescent dye, penetrates all the cells and binds to the AT-rich region of their DNA, imparting a blue fluorescence to the nuclei of both live and dead cells.
In contrast, propidium iodide, a membrane-impermeable fluorescent dye, exclusively enters the dead cells through the damaged portions of their membranes. The dye intercalates between the DNA bases, imparting an additional red fluorescence to the dead cells. The presence of both dyes in the nuclei of these cells make the dead cells appear purple.
Centrifuge the plate at a low speed to settle the cells for enhanced accuracy during the imaging. Image under a fluorescence microscope, and observe two distinct cell populations. Count the number of both cell types.
The percentage of purple cells representing the dead population compared to the total population indicates the cytotoxic potential of the test mitocan.
