The CFSE Distribution Assay is a Powerful Technique for the Analysis of Radiation–Induced Cell Death and Survival on a Single–Cell Level

Background and Purpose: To analyze radiation sensitivity of cells and to monitor cellular responses to irradiation, sensitive test systems for cell death and proliferation on a single-cell level are required. Traditionally, cellular radiation survival is measured using the clonogenic assay as the gold standard. Here it is reported, that labeling of cells with 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester (CFDASE) can be used as a highly sensitive assay to determine cellular response toward irradiation on a single-cell level. Material and Methods: The human malignant cell lines U937 (myelomonocytic, nonadherent), SW48 and SW480 (colorectal, adherent) were labeled with CFDASE, irradiated with either UVB (0-540 mJ/cm 2 ), or X-rays (0-16 Gy). Cell death and proliferation were monitored by cytofluorometry and compared to the clonogenic assay for adherent SW48 and SW480 cells. Results: Dividing nonadherent U937 cells displayed a shift in carboxyfluorescein (CF) fluorescence in parallel with an increased cell count indicating cell proliferation. By comparison, UVB-irradiated U937 cells did not show a shift in CF fluorescence and an increase in cell count indicating cell-cycle arrest. In a mixed cell culture, only the nonirradiated cells divided and concomitantly reduced their fluorescence. Calculating the number of cell divisions it was observed that the nonirradiated cells underwent approximately six cell divisions within 7 days, whereas the irradiated cells divided only once on average. The adherent SW480 colorectal cells showed a more pronounced cell-cycle arrest after irradiation with 240 mJ/cm 2 UVB as compared to cells treated with X-ray up to 16 Gy. Furthermore, the CFSE assay also discriminated colorectal cell lines of different intrinsic radiosensitivities and yielded results comparable to the standard clonogenic assay. Conclusion:Analysis of CF distribution can be employed as a powerful add-on to the clonogenic assay to simultaneously monitor cellular responses toward irradiation on a single-cell level. It constitutes an add-on to the clonogenic assay, especially for nonadherent cells.