Abstract
For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98) were included in the experiment to study its principal and general applicability.
Highlights
Clonogenic and MTT assays are well-known tests for evaluation of chemoradiation studies and radiosensitivity [1,2,3,4]
Clonogenic assay After counting clones, plating efficiency (PE) and survival fraction (SF) can be calculated using the following equations [9]: PE = # of colonies formed × 100%
Multiple MTT assay MTT assay was done once every day with one column of each multi-well-plate to follow up exponential growth
Summary
Clonogenic and MTT assays are well-known tests for evaluation of chemoradiation studies and radiosensitivity [1,2,3,4]. Clonogenic assays are commonly used to investigate survival of irradiated cancer cells, whereas MTT assays are well known to study chemosensitivity [5] or toxicity [6] of drugs in human tumor cell lines. The assay is less common to study survival of cancer cells after irradiation, in particular when the MTT assay is performed for studying proliferation of treated cells. The MTT assay is based on the formation of darkcolored formazan dye by reduction of the tetrazolium salt MTT by metabolically active cells [7]. The water-insoluble formazan dye forms crystals, which can be dissolved in an organic solvent and the amount can be determined semi-automatically
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