MarkerGene™ Live:Dead/Cytotoxicity Assay Kit Green/Red Staining

Product ID: M0795



Unit SizePriceQuantity 
1kit
$247.39
  • Buy 5 for $197.91 each and save 20%

Availability: In stock


Description

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.

Esterases are a family of catabolic enzymes that cleave esters in a wide range of naturally occuring substrates intracellularly. They are ubiquitous to almost all living organisms. Esterase activity can therefore be used to monitor viability of living cells. This kit uses a fluorogenic substrate that, upon cleavage by intracellular esterases, produces a green fluorescent product that is retained intracellularly. Viability and cell number measurements are easily obtained either in vitro, in cell preparations, or in vivo. This kit also contains a red emitting DNA stain that is membrane impermeant to live cells, but can enter the nucleus of dead cells and stain the chromatin (red fluorescence color). Measurement of red versus green fluorescence gives an accurate measurement of dead versus live cell number (viability). The kit contains enough substrate solutions for hundreds of assays and control experiments, and a detailed protocol for use. See the references below for more information and applications.

Technical Data
SKU M0795
Unit Size 1kit
Detection Method Fluorescence

References and Citations

Citations:

  • Olekson M, Faulknor RA, Hsia HC, Schmidt A, Berthiaume F (2015) "Soluble receptor for advanced glycation end products improves stromal cell–derived factor-1 activity in model diabetic environments." Advances in Wound Care.
  • Clay G, Modha S, Schomacker P, Cooper J A. (2015, April). "Development of scaffold for use in osteochondral tissue engineering." In Biomedical Engineering Conference (NEBEC), 2015 41st Annual Northeast(pp. 1-2). IEEE.
  • Fowlkes V, Wilson CG, Carver W, Goldsmith EC. (2013)"Mechanical loading promotes mast cell degranulation via RGD-integrin dependent pathways." J Biomech. 46(4):788-95.
  • Yadav VR, Sahoo K, Awasthi V. (2013) "Preclinical evaluation of 4-[3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl]-4-oxo-2-butenoic acid, in a mouse model of lung cancer xenograft." Br J Pharmacol. 170(7):1436-48.
  • Nooeaid P, Salih V, Beier JP, Boccaccini AR.(2012) "Osteochondral tissue engineering: scaffolds, stem cells and applications." J Cell Mol Med. 16(10):2247-70.
  • Yanez M, Rincon J, Cortez P, Günther N, Boland T, Maria CD (2012) "Printable cellular scaffold using self-crosslinking agents." Journal of Imaging Science and Technology 56(4): 40506-1-40506-5(5).
  • McArdle J, Schafer XL, Munger J. (2011) "Inhibition of calmodulin-dependent kinase kinase blocks human cytomegalovirus-induced glycolytic activation and severely attenuates production of viral progeny." J Virol. 85(2):705-14.
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References:

  • "Direct visualisation and quantification of cellular cytotoxicity using two colour fluorescence." Kroesen, B.J., Mesander, G., ter Haar, J.G., The, T.H., de Leij, L., (1992) J. Immunol. Methods 156:47-54.
  • "Analysis of early apoptotic events in individual cells by fluorescence intensity and polarization measurements." Zurgil, N., Shafran, Y., Fixler, D., Deutsch, M., (2002) Biochem. Biophys. Res. Commun. 290:1573-1582
  • "Flow cytometric assessment of viability of lactic acid bacteria." Bunthof, C.J., Bloemen, K., Breeuwer, P., Rombouts, F.M., Abee, T., Appl. Environ. Microbiol.(2001) 67:2326-2335.
  • "Flow cytometry and cell sorting for yeast viability assessment and cell selection." Deere D, Shen J, Vesey G, Bell P, Bissinger P, Veal D. Yeast 14, 147-160 (1998)
  • "Rapid flow cytometric assay for the assessment of natural killer cell activity." Chang, L., Gusewitch, G.A., Chritton, D.B., Folz, J.C., Lebeck, L.K., Nehlsen-Cannarella, S.L., (1993) J. Immunol. Methods. 166:45-54.
  • "Dehydrothyrsiferol does not modulate multidrug resistance-associated protein 1 resistance: a functional screening system for MRP1 substrates." Pec, M.K., Aguirre, A., Fernandez, J.J., Souto, M.L., Dorta, J.F., Villar, J.. Int J. Mol. Med. 10:605-608.
  • "Improved microplate fluorometer counting of viable tumor and normal cells." Riordan, H.D., Riordan, N.H., Meng, X., Zhong, J., Jackson, J.A., Anticancer Res. (1994) 14:927-931.
  • "Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product." Breeuwer P, Drocourt JL, Bunschoten, Zwietering MH, Rombouts FM, Abee T (1995) Appl. Environ Microbiol. 61(4): 1614-1619.
  • "Rapid viability assessment of yeast cells using vital staining with 2-NBDG, a fluorescent derivative of glucose." Oh KB, Matsuoka H (2002)Intl. J. Food Microbiol. 76(1-2): 47-53.
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Technical Support

Question about this product? Ask a Scientist!

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:


⚛ Question: How well does propidium iodide pick up dead cells? If they are just injured, can it detect these cells also?

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.

⚛ Question: Is there a better more detailed protocol for the usage of these reagents? The one that's online does not provide much info on the application to my instrument.

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.

⚛ Question: Can I co-stain mitochondrial membranes at the same time as staining with this kit?

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.
⚛ Question: I want to use this kit for primary neurons using PI staining for fluorescence microscopy. Could you tell me if there is a need to fix the cells before using your kit? And in general is it possible to fix the cells before using the kit?

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.