11465015001

Roche

Cell Proliferation Kit II (XTT)

Synonym(s):
xtt
Pricing and availability is not currently available.

Quality Level

form

liquid

usage

sufficient for ≤2,500 tests

packaging

pkg of 1 kit

mfr. no.

Roche

storage condition

protect from light

application(s)

cell analysis: suitable
detection: suitable
tissue culture: suitable

λmax

450-500 nm

detection method

colorimetric

shipped in

dry ice

storage temp.

−20°C

General description

The Cell Proliferation Kit II (XTT) is a colorimetric assay for the nonradioactive quantification of cellular proliferation, viability, and cytotoxicity. Sample material is either adherent or suspension cells cultured in 96-well microplates.

Colorimetric assays analyze the number of viable cells by the cleavage of tetrazolium salts added to the culture medium. This technique requires neither washing nor harvesting of cells, and the complete assay, from microculture to data analysis by an ELISA reader, is performed in the same microplate.
More recently, the tetrazolium salt XTT was described. In contrast to MTT, the cleavage product of XTT is soluble in water; therefore, a solubilization step is not required. The tetrazolium salt XTT is cleaved to formazan by a complex cellular mechanism. This bioreduction occurs in viable cells only, and is related to NAD(P)H production through glycolysis. Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.

Application

The Cell Proliferation Kit II (XTT) is used for the nonradioactive, spectrophotometric quantification of cell proliferation and viability in cell populations using the 96-well-plate format. It can be used for:
  • Measurement of cell proliferation in response to growth factors, cytokines, mitogens, and nutrients.
  • Analysis of cytotoxic and cytostatic compounds, such as anti-cancer drugs and other pharmaceutical compounds.
  • Assessment of growth-inhibitory antibodies and physiological mediators that inhibit cell growth.
  • Testing of biocompatibility of various scaffolds, employed in bone tissue engineering, for bone cell growth.
  • cell viability assay.

Features and Benefits

  • Safe and easy: Eliminate radioactive isotopes, washing steps, and additional reagents.
  • Accurate: The absorbance obtained strongly correlates to the cell number.
  • Sensitive: Detect low cell numbers.
  • Fast: Process a large number of samples using a multi-well ELISA reader.

Packaging

1 kit containing 2 components.

Principle

The assay is based on the cleavage of the tetrazolium salt XTT in the presence of an electron-coupling reagent, producing a soluble formazan salt. This conversion only occurs in viable cells. Cells grown in a 96-well tissue culture plate are incubated with the XTT labeling mixture for 2 - 20 hours. After this incubation period, the formazan dye formed is quantitated using a scanning multi-well spectrophotometer (ELISA reader). The measured absorbance directly correlates to the number of viable cells.
Cell proliferation and viability assays are of particular importance for routine applications in cell biology. Tetrazolium salts (e.g., MTT, XTT, WST-1) are particularly useful for this type of analysis. Tetrazolium salts are cleaved to formazan by the succinate-tetrazolium reductase system (EC 1.3.99.1) which belongs to the respiratory chain of the mitochondria, and is only active in metabolically intact cells.

Preparation Note

Working solution: Preparation of solutions
Thaw XTT labeling reagent and electron-coupling reagent, respectively in a water bath at 37 °C. Mix each vial thoroughly to obtain a clear solution.
XTT labeling mixture
To perform a cell proliferation assay (XTT) with one microplate (96 wells) mix 5 ml XTT labeling reagent with 0.1 ml electron coupling reagent.
Note: To obtain reliable results thaw and mix XTT labeling reagent and electron coupling reagent immediately before use.
Working instruction
Cells are grown in microplates (tissue culture grade, 96 wells, flat bottom) in a final volume of 100 μl culture medium per well, according to the media needs of the cells in a humidified atmosphere (e.g., 37 °C, 6.5% CO2).
The incubation period of the cell cultures depends on the particular experimental approach and on the cell line, used for the assay. For most experimental setups, the incubation of cells for 24 to 96 hours is appropriate.
After the incubation period, add to each well 50 μl of the XTT labeling mixture, prepared as described above (final XTT concentration 0.3 mg/ml).
Incubate the microplate for 4 to 24 hours in a humidified atmosphere (e.g., 37 °C, 6.5% CO2).
Note: The incubation time varies with the individual experimental setup (e.g., cell type and cell concentration, used). Therefore, we recommend to measure the absorption as described at different time points after addition of XTT labeling mixture (e.g., 4, 6, 8, 12, and 18 hours) using one and the same microplate to determine the optimal incubation period for the particular experimental setup.
Storage conditions (working solution): Thaw reagents immediately before use. It is recommended to prepare appropriate aliquots 5 ml XTT labeling reagent and 0.1 ml electron coupling reagent are required for the performance of the assay with one microplate (96 wells)
Note: Avoid repeated thawing and freezing.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

Product No.
Description

  • XTT Labeling Reagent

  • Electron-coupling Reagent

RIDADR

NONH for all modes of transport

WGK Germany

nwg

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Certificate of Analysis
Certificate of Origin
Eun Young Park et al.
The Journal of biological chemistry, 289(13), 9254-9262 (2014-02-12)
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A combination of photodynamic therapy and chemotherapy displays a differential cytotoxic effect on human metastatic melanoma cells.
Nsole Biteghe F A and Davids L M
Journal of Photochemistry and Photobiology. B, Biology, 166, 18-27 (2017)
High-Temperature Requirement A1 (Htra1) - A Novel Regulator of Canonical Wnt Signaling.
Oriane G, et al.
Scientific Reports, 7(1), 17995-17995 (2017)
Senescent growth arrest in mesenchymal stem cells is bypassed by Wip1-mediated downregulation of intrinsic stress signaling pathways.
Lee J S, et al.
Stem Cells, 27(8), 1963-1975 (2009)
Helle Lysdahl et al.
BioResearch open access, 3(6), 278-285 (2014-12-04)
Clinical trials using bone morphogenetic protein-2 (BMP2) for bone reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect...
Articles
Cell based assays for cell proliferation (BrdU, MTT, WST1), cell viability and cytotoxicity experiments for applications in cancer, neuroscience and stem cell research.
Read More
Protocols
XTT assay protocol for measuring cell viability, proliferation, activation and cytotoxicity. Instructions for XTT reagent preparation and examples of applications.
Read More

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