The radiation survived NSCLC cells express cancer stem cell markers. A549 and H460 cells were irradiated (5Gy) and cultured in adherent conditions for a week. Cells were collected and plated as single cell suspensions, in stem-cell selective conditions, to form floating tumor spheres. To obtain adherent radiation survived cells, cells were kept in the plates for two weeks after IR-treatment. Expressions of markers were tested in non-irradiated cells, adherent cells that survived IR treatment, non-irradiated sphere cells, and sphere cells that survived irradiation. (A) Morphology of adherent cells that survived IR treatment and spheres developed from IR-treated cells are shown. (B) Radiation survived cell populations are enriched in the tumor-sphere forming cells. Non-irradiated cells and radiation survived cells were harvested, and transferred into the stem cell selective conditions. 10 days later the tumor spheres, from the first generation, were counted under the microscope. The tumor spheres, from the first generation, were used to develop the tumor spheres for the second generation. The second generations of tumor spheres were used to develop the third generation spheres used for further study. (C-F) Analysis of CD44, CD24 and CD166 expression. Cells in 96-well plates were fixed, incubated with the respective antibody and stained with Hoechst33342. Cell images were acquired using the ArrayScan HCS Reader (40× objective) and analyzed using the Target Activation BioApplication Software Module. (C) The representative images of the A549 radiation survived sphere cells stained for CD44 and CD166 are shown. (D-F) The total average fluorescence intensities of CD24 (D), CD44 (E) and CD166 (F) in the non-irradiated cells (grey), IR- survived adherent cells (green), non-irradiated sphere cells (red) and in the IR-survived sphere cells (blue) are presented. The fluorescence intensities of respective IgG controls were subtracted. Each point presents average intensities (pixels) estimated for 3000 cells.