MarkerGene™ Live:Dead/Cytotoxicity Assay Kit Green/Red Staining
Product ID: M0795
This kit allows fast and easy measurement of both living and dead cells by measuring intracellular esterase activity (live cells) with a green emitting dye while staining of nucleic acids in chromatin (dead cells) with a red emitting dye.
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We pride ourselves on the high quality of our products and want you to get the best possible results from your assays. If you have any questions about this product or need help optimizing your protocol check out the product FAQs below or ask your own question and one of our expert scientists will get back to you asap:
The assay principle is based on the fact that propidium iodide cannot penetrate the membranes of live cells. So there should not be any PI staining of cells that are still viable. One of the definitions of cell death is membrane disruption and depolarization, whereby the PI can enter the cells, bind to the chromatin and change fluorescence color staining them red.
But, of course, there can be some cells that are essentially dead (so called "ghosts") whose membranes have not yet been compromised for some reason, and therefore will not come up as dead in this method. But from what I am told, the usual levels of these cells is low ( i.e. < 5% or so). Live cells turn over the FDA substrate, staining live cells green, but when the cell membrane is compromised, this green dye will leak out and they will not be green any longer. Certainly, a clonal assay is the best method to precisely measure cell viability, especially if you are examining cytotoxicity from drug application. But our live:dead kit has been shown to match clonal data quite well.
Unfortunately, we don't give specific information about individual instruments or those from different manufacturers, since these instruments often change or updated. I would suggest instead that you contact the manufacturer of your particular instrument for information about setting up the filters, and running the particular assays. Our product information sheet is provided as a general method for people using many different types of instruments.
I would suggest using instead our product M1076: 10-Octadecylacridine orange bromide for costaining mitochondiral membranes. This dye has been used to analyze mitochondria by flow cytometry, characterize multidrug resistance, and measure changes in mitochondrial mass during apoptosis.
Here are some references for the protocol for staining. You should be able to add the 10-Octadecylacridine orange directly to the cell staining solutions (@ ~ 20 ug/mL concentration) to counterstain.
- Ratinaud MH, Leprat P, Julien R. (1988) "In situ flow cytometric analysis of nonyl acridine orange-stained mitochondria from splenocytes."Cytometry 9: 206.
- Maftah A, Petit JM, Julien R. (1990) "Specific interaction of the new fluorescent dye 10 N-nonyl acridine orange with inner mitochondrial membrane. A lipid-mediated inhibition of oxidative phosphorylation."FEBS Lett. 260: 236. Kessel, D., (1991) "Characterization of multidrug resistance by fluorescent dyes." Cancer Res. 51: 4665.
- Cossarizza, A., (1994) "Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level." Exp. Cell Res. 214: 323.
In general, the assay is designed to be used with live cells, and the fluorescence read pretty much immediately after staining.
It is not possible to use this assay technique with fixed cells, since the dye (propidium iodide) for "dead" cells works by measuring cell membrane integrity, and fixing the cells will make all of the cell look like they are "dead".
It is fine to use the Moviol 4-88 mounting medium or Gel/Mount for preparing slides, but we typically just use a drop of media and place the cover slip onto the slide inverted for viewing. Alternately, and more typically, we run the assay in 12- or 96-well tissue culture plates and measure the fluorescence from the two dyes at the two wavelengths using a microtiterplate reader. But you might want to see the results, at least the first time, using your fluorescence microscope technique, to make sure it is working properly.