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Protocol no. 43
COLORIMETRIC CYTOTOXICITY ASSAYS FOR ANCHORAGE DEPENDENT CELLS

The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere. Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays.

CONTACT

Dr. Margaret E. Watts
Cancer Research Campaign - Gray Laboratory P.O. Box 100
Mount Vernon Hospital Northwood Middlesex, HA6 2JR UK
Tel: England - 092 7428611, ext 234

RATIONALE

A simple cell culture-based cytotoxicity system has been modified so as to enable a method for determining the potential toxicity of compounds under hypoxic conditions. Furthermore, the use of colorimetric assays to determine cell survival has permitted the procedure to become highly automated, thus yielding a rapid test system. The assays are: MTT assay The tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is taken up into cells and reduced by a mitochondrial dehydrogenase enzyme to yield a purple formazan product which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. Solubilisation of the cells results in the liberation of the product which can readily be detected using a simple colorimetric assay. The ability of cells to reduce MTT provides an indication of the mitochondrial integrity and activity which, in turn, may be interpreted as a measure of viability and/or cell number. Methylene blue assay The total number of cells present in a culture may be determined, regardless of their viability/integrity, using this method. Cells are simultaneously fixed and stained in a solution of dye in ethanol. After rinsing the dye is liberated and solubilised, the exact amount being determined colorimetrically. The amount of dye provides an indication of cell number. Cells are exposed to test compounds and subsequently cultured. At the end of the culture period the number of cells/cell growth is determined by either of the two assays mentioned above. Comparison of results between control and test cultures provide an indication of the cytotoxic effect of test compounds.

BASIC PROCEDURE

Cells are incubated under hypoxic conditions with a range of drug concentrations. Samples of cells are removed and aliquots are added to 96-well plates and cultured until the control cells reach 80-90% confluence. Cell growth/survival is assessed using one of the two colorimetric assays. MTT assay MTT solution is added to each well and the plates incubated. The medium is aspirated, replaced with DMSO and the plates agitated. The absorbance is read for each well and the surviving fraction calculated. Methylene Blue assay The medium is aspirated and methylene blue solution is added to each well and incubated. The stain is aspirated and the plates rinsed. Sarkosyl solution is added to each well and the plates agitated. The absorbance is read for each well and the surviving fraction calculated.

CRITICAL ASSESSMENT

This is a simple and rapid method which may be used as a screening assay for compounds which are cytotoxic under hypoxic conditions. The use of 96-multiwell plates with 8-12 channel pipettes, microplate washer and shaker together with a plate reading spectrophotometer enables large numbers of plates to be processed quickly (especially if interfaced with a computer). One limitation to the use of this assay for screening large numbers of hypoxic cell radiosensitisers and hypoxic-specific cytotoxins is the possibility of the plastic-ware used in the procedure occluding oxygen at radiobiologically significant levels. All work during the exposure to test compound phase of the procedure should, therefore, be carried out in glass flasks before the transfer stage to multiwell plates. Attempts have been made to overcome the problem by using long periods of hypoxia with high cell densities and small volumes with plastic multiwell plates. These factors, however, counteract the advantages of a rapid and simple assay system. The choice of cell number initially plated into the 96-well plates should be determined such that the control cells undergo 8-9 divisions during the incubation period before reaching 80-90% confluent. This number of cells should be sufficient to enable detection of cell death and growth inhibition effects. If larger cell numbers and shorter assay times are used the cultures rapidly become confluent and cells destined to die as a result of the toxic effect of a test compound may still be metabolically active at the point where cell number is estimated, thus resulting in overestimation of survival and an underestimation of the toxic potential of the compound. Colorimetric assays which are suitable for automation have a number of advantages over conventional clonogenic assays. Primarily they provide a relatively sensitive, reproducible, rapid means of determining the cytotoxic potential of large numbers of chemical/compounds. There are some uncertainties, however, regarding absorbance readings at low values. This means that measurement of cell survival when the survival fraction or cell number is very low is much less likely to be accurate (e.g. fractions below 0.05). Although there is reasonable agreement between colorimetric and clonogenic assays for surviving fractions down to about 0.05, only the clonogenic assay results can be confidently interpreted at levels below this value (clonogenic assays can measure survival down to 10-4 surviving fraction). Choice of cell line The V79-379A cell line was selected because it is widely used in radiobiological research. The two human tumour cell lines, LoVo and HT-1080 were chosen as representative human tumour cell lines which would grow as colonies in a relatively simple medium. For this protocol the cells were used under hypoxic conditions to mimic the conditions that occur in many tumours. However, both the MTT and MB assays can be used for measuring the survival of cells after exposure to test conditions of any kind, including aerobic conditions. In a separate series of experiments the MB assay has been used for measuring survival of endothelial cells which although anchorage dependent are migratory in nature and so do not form colonies. There is a reasonable agreement between MTT and MB assays. MTT assay Formation of the formazan product has been found to correlate well with cell number, although not always in a strict linear fashion, the assessment of results must, therefore, be carefully interpreted. If surviving fraction is calculated directly from the ratio of absorbances an estimation, not an absolute value, of cell survival will occur. Calibration curves may be constructed for each experiment, but this results in the assay becoming more time consuming, thus reducing its advantage over conventional clonogenic assays. It compares favourably with several other methods used in the determination of cell number/viability e.g. dye exclusion. It is a rapid, sensitive, relatively simple assay to perform which lends itself to semi-automation. The MTT assay has several drawbacks: The MTT assay is not readily adaptable for use with static cell populations or those of low mitochondrial activity. Certain compounds may selectively affect the mitochondria of the cells resulting in a greatly overestimated/underestimated level of toxicity. Different cell lines are likely to give different absorbance levels when at similar degrees of confluence. MTT is mutagenic and, therefore, must be handled with care. Once the assay is initiated it must be completed as the coloured formazan product deteriorates over time. MB assay MB is less hazardous to work with than certain other dyes or compounds e.g. MTT, used in cell survival assays. The assay is relatively short as no long periods of incubation are required. The plates do not have to be read immediately but may be dried after the cells are fixed and exposed to the dye. The process may be completed at a later date.

TEST STATUS

In-house
ORGANISATIONS USING THE TEST
None

CHEMICALS TESTED

Misonidazole
Pimonidazole

REFERENCES

  1. Finlay, G.J., Baguley, B.C. & Wilson, W.R. (1984) A semi-automated microculture method for investigating growth inhibitory effects of cytotoxic compounds on exponentially growing carcinoma cells. Anal. Biochem., 139, 272-277.
  2. Slater, T.F., Sawyer, B. & Strauli, U. (1963) Studies on succinate-tetrazolium reductase systems. III Points of coupling of four different tetrazolium salts. Biochem. Biophys. Acta., 77, 383-393.
  3. Watts, M.E., Roberts, I.J. & Woodcock, M. A comparison of colorimetric and clonogenic assays for hypoxic-specific toxins with hamster and human cells. Int. J. Radiation Oncology Biol. Phys., 16, 939-942.

IP-43 © December 1990