Generi Biotech - Compounds and Services for Molecular Biology

• Quantitative PCR
  • Sets for Gene Expression
  • qPCR sets for reference genes
  • Custom-Made qPCR Sets
  • Probe Master Mix
  • Standards for qPCR
  • Internal Positive Controls
• Sequencing
  • Standard Sequencing
  • 16S/ITS Sequencing
• Molecular Cloning
  • Custom cloning
  • Plasmid Amplification and Isolation
  • Competent Bacteria
• Cell Culture Applications
  • Recombinant Proteins
  • Recombinant Human SHBG
  • Cytotoxicity Testing
  • Luminescence Solution
  • Mycoplasma Detection
• Biological Identification
  • Cell Line Authentication
  • Paternity Outsourcing

XTT test

The method is based on the ability of metabolically active cells to convert yellow tetrazolium salt XTT into orange formasan dye. As this conversion occurs only in live cells, cytotoxicity of various chemical substances can be determined in this way. The originating formasan dye is soluble in water and can be directly quantified using any classical spectrophotometer (ELISA reader). Complex rinsing is thus avoided, which is necessary, for example, in the older MTT method. This guarantees high accuracy of XTT test and allows for fast processing of the data obtained.

Example

Hep2, MCF-7 and HepG2 cells were applied onto the 96-well plate into appropriate medium as follows: 2 x 10⁴ (Hep2), 3 x 10⁴ (MCF-7) or 5 x 10⁴ (HepG2) cells per well. Upon achieving 80% confluence, the medium was replaced and etoposide (ETO) was added in appropriate concentrations, and/or DMSO as a positive control. The cells were incubated with etoposide for 72 hours. Then the XTT test was performed. Absorbance values reduced by the positive control value are expressed as percentage of viability of the cells related to the negative control sample (uninfluenced cells).

Fig. Etoposide (ETO) cytotoxicity in cell lines Hep2, MCF-7 and HepG2 evaluated using XTT test. The data were processed in the program GrafPad Prism, version 4 (GraphPad Software, San Diego, CA, USA). IC₅₀ values correspond to etoposide concentration under which the cell viability is equal to 50%. N = 2; n = 3.

References

• Eskiocak, U., O. D. Iseri, et al. (2008). "Effect of doxorubicin on telomerase activity and apoptotic gene expression in doxorubicin-resistant and -sensitive MCF-7 cells: an experimental study." Chemotherapy 54(3): 209-16.
• Jost, L. M., J. M. Kirkwood, et al. (1992). "Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells." J Immunol Methods 147(2): 153-65.
• Khot, P. D., P. A. Suci, et al. (2008). "Candida albicans viability after exposure to amphotericin B: assessment using metabolic assays and colony forming units." J Microbiol Methods 72(3): 268-74.
• Mowat, E., S. Lang, et al. (2008). "Phase-dependent antifungal activity against Aspergillus fumigatus developing multicellular filamentous biofilms." J Antimicrob Chemother
• Scudiero, D. A., R. H. Shoemaker, et al. (1988). "Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines." Cancer Res 48(17): 4827-33.