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Rapid Fluorescence-based Assay for Radiosensitivity and Chemosensitivity Testing in Mammalian Cells in Vitro

Department of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

An efficient and rapid cytotoxicity assay has been developed, particularly for radiobiological studies, utilizing 96-well microtiter plates. Several days after treatment, cell numbers per well were measured by fluorescent intensity using an automatic reader after staining with the DNA specific dye Hoechst 33258. For radiobiological applications, a microtiter plate irradiation box was designed and built which allowed a variable number of wells (minimum 4, maximum 16) to be irradiated at one time. In this manner, complete dose-response curves could be obtained from one plate. The assay depends on the growth of surviving and untreated cells, and by appropriate choice of conditions (cell numbers plated, time of assay), cell survival curves for this quick fluorescence assay were in reasonable agreement with those from a clonogenic assay for cisplatin and X-ray-induced cell killing. The assay can span 1.5–2 decades of cell survival and is suitable for any cell line which grows as a monolayer.

Radiobiological applications were tested using agents or conditions which modified radiation damage. Firstly, sublethal damage repair could be demonstrated in RIF1 mouse tumor cells by comparing the survival curve for a single X-ray dose with that for two fractions separated by 4 h. Secondly, incorporation of 5-iodo-2'-deoxyuridine into cellular DNA was shown to radiosensitize Chinese Hamster cells, with similar enhancement ratios obtained from the fluorescence and clonogenic assays. Thirdly, radiosensitization by cislatin and radioprotection by cysteamine could be readily measured using the quick fluorescence assay. The ability to have multiple dose groups per plate makes it an efficient assay for both radiosensitivity and chemosensitivity testing.

Received 6/13/88. Revised 9/21/88. Accepted 10/14/88.

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