Rapid fluorescence ratio assay for detecting changes in radiosensitivity.
To test modifications in sensitization to radiation or drugs in preclinical studies of cancer therapy, the colony-forming assay is regarded as the gold standard. Because this assay is time consuming, somewhat laborious, and unsuitable for rapid screening, development of other assays is desirable. We describe here an assay based on the detection of enhanced green fluorescence protein (EGFP) with flow cytometry that is particularly suitable for genetic manipulation studies in which the gene of interest is introduced together with EGFP as reporter. It is easily adaptable to other reporters, however, whether naturally fluorescent or requiring immunochemical staining. Cells are irradiated as mixed populations of a known standard cell line (nonfluorescent) together with the genetically manipulated cell line expressing EGFP. Ratios of fluorescent and nonfluorescent cells are measured before treatment and several days after treatment. If the cell populations have equal radiosensitivities, the ratio remains unchanged. Changes in the ratio indicate changes in radiosensitivity. The assay was validated for two situations in which dominant negative peptides inhibiting DNA repair were expressed in A549 human lung cells and affected radiosensitivity.
Hofland
I
I
Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Ramakers
B
B
Begg
A C
AC
Vens
C
C
D016923:Cell Death / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D011836:Radiation Tolerance; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019951:DNA Polymerase beta