A modified computer-assisted colorimetric microtitre assay (MTT) to assess in vitro radiosensitivity of V79, CaSki, HeLa and WiDr cells.
The non-clonogenic MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, was modified and a new procedure of analysis is proposed. The colorimetric assay could be performed semi-automatically using microtitre plate reader connected to a personal computer. Data are processed and plotted with a customized program. To ensure that both control and irradiated microtitre plates contain exponentially growing cells at the time of analysis, the calculation of relative survival is based on a series of well-defined cell numbers initially seeded. To make the two endpoints of non-clonogenic and clonogenic assays comparable, i.e. counting of living cells versus counting of colonies, radiation-induced progression delay was incorporated into the calculation. Radiation-induced cell killing and progression delay could be determined in a single analysis, but in an independent way. X-ray survival curves were generated for V79, CaSki, WiDr and HeLa cells using the non-clonogenic and a standard clonogenic assay. Using the linear-quadratic formula, the resulting parameters alpha, beta and the mean inactivation dose were not significantly different. The described assay is a feasible and reproducible technique for determination of cellular survival, which may be able to incorporate progression delay. The equivalence to a clonogenic survival assay could be proven.
Eble
M J
MJ
Department of Radiotherapy, University of Heidelberg, Germany.
Hensley
F W
FW
Flentje
M
M
Schlotz
A
A
Wannenmacher
M
M
D000818:Animals; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured / Q000528:radiation effects*; D002999:Clone Cells; D003124:Colorimetry; D006224:Cricetinae; D004307:Dose-Response Relationship, Radiation; D005562:Formazans / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010088:Oxidoreductases / Q000378:metabolism; D011834:Radiation Monitoring / Q000379:methods; D014965:X-Rays
D005562:Formazans; D010088:Oxidoreductases