RAD51-GFP as a live cell marker. Panel a: RAD51-GFP foci in live SiHa cells stably transfected with RAD51-GFP are shown 24 hours after exposure to 8 Gy. The phase contrast image is indicated by red outlines. Panel b: Co-localization between RAD51-GFP and anti-RAD51 antibody staining (red) in an untreated SiHa cell. Panel c: Co-localization between RAD51-GFP (green) and γH2AX antibody staining (red) in SiHa cells 24 hours after exposure to 8 Gy. Panel d: Analysis of the fraction of SiHa-RAD51-GFP cells that develop foci as a function of time after exposure to 4 Gy or 8 Gy. Results from several experiments are pooled. The dotted line and open circles show the development of RAD51 antibody-labelled foci after exposure to 4 Gy. The single open triangle shows the fraction of RAD51 antibody-labeled cells 24 hours after 8 Gy. Panel e: The fraction of cells that exhibit RAD51-GFP foci or γH2AX foci 24 hours after exposure to radiation, measured microscopically. Panel f: SiHa-RAD51-GFP cells expressing high levels of RAD51-GFP after 24 hours after irradiation were sorted on the basis of GFP, fixed and stained for γH2AX. The average intensity of the populations was measured using flow cytometry. The mean and standard error for 3 sorted populations is shown. Panel g: Clonogenicity of SiHa-RAD51-GFP cells after 0 or 3 Gy exposure. Cells in 8-well dishes were irradiated and 24 hours later, wells containing one or two doublets were scored for the presence or absence of γH2AX foci (daughter cell pairs show the same foci patterns). Dishes were returned to the incubator for 2 weeks to form colonies. The fraction of doublets with foci that survived treatment or the fraction lacking foci that survived treatment was calculated.