Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

Clonogenic and MTT assays are well-known tests for evaluation of chemoradiation studies and radiosensitivity [1-4]. 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 aim of this study is to compare the well-established clonogenic assay with an adapted version of the MTT proliferation assay to overcome limitations such as long duration of experiment, low sample throughput, limited error level and time-consuming counting of clones.The MTT assay is based on the formation of dark-colored formazan dye by reduction of the tetrazolium salt MTT by metabolically active cells [7]. After some incubation, the water-insoluble formazan dye forms crystals, which can be dissolved in an organic solvent and the amount can be determined semi-automatically using a microplate reader. Absorbance readings are related to the number of cells [8] therefore providing the possibility to use the MTT assay as a proliferation assay to assess cell growth after irradiation. In the present study our adapted version of the MTT assay is compared to the clonogenic assay in order to open the possibility of replacing one by the other.In the literature, several studies on comparability of MTT and clonogenic assay can be found. There, the MTT assay is done as a single-point assay after a defined time following treatment. In this case, much information about the growth behavior of the cells is lost (doubling time, lag phase, growth behavior etc.). In contrast, with our multiple MTT assay, we collect all those data and use them for more detailed interpretation.Experiments were carried out with A 549 cells (human NSCLC cell line), LN 229 cells (human glioblastoma cell line) and F 98 cells