Quantitative assessment of cell viability

Measurement of viable cells through metabolic activity.

The XTT test is an in vitro cytotoxicity assay based on the cleavage of the yellow tetrazolium salt XTT into an orange, water-soluble formazan product by dehydrogenase activity in active mitochondria.

A decrease in the number of viable cells results in reduced mitochondrial dehydrogenase activity within the sample. This reduction directly correlates with the amount of orange formazan formed, which is measured by absorbance.

Using the XTT assay, cell viability and proliferation following treatment with the test item are determined colorimetrically.

Assessment of mitochondrial dehydrogenase activity by extraction method and XTT dye

  • The test item is extracted under agitation for a defined period in DMEM supplemented with 10% FBS at 37 ± 1°C, and L929 cells are incubated with the extract.
  • At the end of the incubation period, the cells are evaluated microscopically and the XTT reagent and electron coupling reagent are added.
  • Cells are incubated for a further 1 to 2 hours, and absorbance is measured at 450 nm (reference wavelength 650 nm).
  • Dehydrogenase activity below 70% compared with untreated control cultures (solvent control) is considered a clear cytotoxic effect according to ISO 10993-5.

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XTT test protocol

Protocol

Cell line

L929 cells (ATCC No. CCL1, NCTC clone 929; connective tissue mouse cell line, clone of strain L, DSMZ)

Analysis

The XTT test is based on the cleavage of the yellow tetrazolium salt XTT to form an orange water-soluble formazan product by dehydrogenase activity in active mitochondria

Concentrations

4 extract concentrations: 29.6%, 44.4%, 66.7% and 100%

Extraction time

4 - 72 h at 37 ± 1°C

  • 4 h (short-term contact and intact skin or mucosa)
  • 24 ± 2 h (short-term and limited contact to patient)
  • 72 ± 2 h (prolonged and permanent contact)

Incubation time

24 - 72 h at 37 ± 1°C

Quality controls

Solvent Control: DMEM 10% FBS

Negative control: Polypropylene extracted in DMEM 10% FBS

Positive control: Latex extracted in DMEM 10% FBS

Data delivery

Mitochondrial dehydrogenase activity is determined through the absorbance at 450 nm (reference wavelength 650 nm)

Positive prediction

Dehydrogenase activity of less than 70% compared to untreated control cultures (solvent control) is considered as a clear cytotoxic effect

 

References

  1. Scudiero, P. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Thierney, S.; Nofziger, T. ; Currens, M. J.; Seniff, D.; Boyd, M. R.: 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, 4827 – 4833
  2. ISO 10993-5: 2009, “Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity“
  3. ISO 10993-12: 2012, “Biological evaluation of medical devices – Part 12: Sample preparation and reference materials“