MarkerGene™ Fluorescent Cellular Senescence Microtiterplate Assay Kit

Product ID: M1405



Unit SizePriceQuantity 
1 Kit
$328.64
  • Buy 5 for $262.91 each and save 21%

Availability: In stock


Description

Uses the high-sensitivity substrate fluorescein di-β-D-galactopyranoside (FDG) to quantify senescent cells.

Recent studies have shown that β-galactosidase histochemical staining at pH 6 is a useful enzymatic marker of cellular senescence (the so-called senescence associated beta-galactosidase; SA-β-Gal). While cells entering senescence show this enzymatic activity, immortalized cell types, including tumor or other quiescent cells, are not stained under these conditions. The implications of these methods in such diverse areas as age-related pathology research, tumor analysis and tissue culture maintenance is significant. FDG turnover produces the highly fluorescent product fluorescein, which is easily detected using an appropriate filter set for fluorescein fluorescence (i.e. EX 485nm and EM 535 nm) in a microtiterplate assay format. This assay kit contains all of the reagents for performing up to 100 assays using 6-well or 12-well tissue culture plate samples of cells or for cells that have been grown onto sterile cover slips for analysis from other plate sizes. In addition, the kit also contains reaction buffers, dilution solvents, a detailed protocol and references for use.

See also product Product M0250 for additional information and references.


Technical Data
SKU M1405
Unit Size 1 Kit
Detection Method Fluorescence

References and Citations

Citations:

  • Forouzandeh F, Salazar G, Patrushev N, Xiong S, Hilenski L, Fei B, Alexander RW. (2014) "Metformin beyond diabetes: pleiotropic benefits of metformin in attenuation of atherosclerosis." J Am Heart Assoc. 3(6):e001202.
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References:

  • Dimri GP, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens m, Rubeli I, Pereira-Smith O, Peacocke M, Campisi. (1995) "A biomarker that identifires senescent human cells in culture and in aging skin in vivo." PNAS 92: 9363-9367.
  • Yang NC, Hu ML. (2004) "A fluorimetric method using fluorescein di-β-D-galactopyranodise for quantifying the senescence-associated β-galactosidase activity in human foreskin fibroblast Hs68 cells." Anal Biochem 325: 337-343.
  • Eriko M, Nakabayashi K, Suzuki T, Kaul SC, Ogino H, Fujii M, Mitsui Y, Ayusawa D. (1999) "5-Bromodeoxyuridine induces senescence-like phenomena in mammalian cells regardless of cell type or species." J. Biochemistry 126: 1052-1059.
  • Chen JH, Ozanne SE, Hales CN, (2007) "Methods of Cellular Senescence Induction Using Oxidative Species." In "Biological Aging: Methods and Protocols" Methods in Molecular Biology 371: 179-189.
  • Katakura Y, Miura T, Uehara N, Nakata E, Shirahata S (1999) "Senescence Induction in Cancer Cells." In Animal Cell Technology: Challenges for the 21st Century: Proceedings of the joint international meeting of the Japanese Association for Animal Cell Technology (JAACT) and the European Society for Animal Cell Technology (ESACT) 1998, Kyoto, Japan, Part XI, pages 279-282.
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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: If I want to directly grow cells and fix cells in 96-well plate, how much fixing solution and staining solution should I add? In your step 5 and 6, it said to use 500 uL fixing and staining buffer to each well. But this certainly won't fit in 96-well pla
➸ Answer:
The 500ul stated in the protocol is for when 12 well plates are used, for 96 well plates this volume would be reduced to 100ul.
Question: Could we mix the staining buffer and FDG according to the ratio first, then add into the well at one time?
➸ Answer:
It should be fine to mix both the staining buffer and the FDG and add them at the same time. If you are making a large quantity of this mixture, you might want to cool down the staining buffer (ice-bath, for example) prior to adding the FDG solution as this will limit any potential hydrolysis. But after that you can just do things at room temp. per the protocol.
Question: What can be used as a positive control for this kit?
➸ Answer:
The usual controls (hydrogen peroxide or t-Butyl peroxide at non lethal levels) seemed not be appropriate for this fluorescence based assay, probably because of the substrate used in the kit to measure SA-bGal activity, Fluorescein di-Galactopyranoside (FDG), is not stable under the peroxide conditions of that method and breaks down non-specifically.
However, when we tested the kit using well characterized senescent cells obtained from collaborators, the staining patterns for X-Gal and FDG (SA-b-Gal) were very similar (see the data contained in the product information sheet provided in the kit).
We searched for other methods to provide a positive control (inducer of senescence) reagent to add to the kit, but without success. However, we feel confident that the FDG method can measure SA-bGal levels in senescent cells according to the methods described.
To date we have not been able to find a suitable positive control (reagent) to artificially induce senescence in cells lines for the kit, and therefore suggest verifying staining patterns by separate X-Gal staining.