In vitro Cytotoxicity Assay with XTT Dye

In vitro Cytotoxicity Assay with XTT Dye

(mouse cell line L929)

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 the active mitochondria.

A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of the orange formazan formed, as monitored by the absorbance.

With the XTT test, cell proliferation and viability of the cells after treatment with the test item are determined colorimetrically.

XTT Dye

Assessment of mitochondrial dehydrogenase activity by extraction method and XTT Dye

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

Protocol
Cell line	L929 cells (ATCC No. CCL1, NCTC clone 929 (connective tissue mouse), 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 concentrations of the test extract: 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

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
ISO 10993-5: 2009, “Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity“
ISO 10993-12: 2012, “Biological evaluation of medical devices – Part 12: Sample preparation and reference materials“