Radiation- survived sphere cells display upregulation of CXCR4 and PDGFR-beta receptors. Bulk NSCLC cells, radiation survived adherent cells, non-irradiated sphere cells from the third generation, and radiation survived sphere cells were seeded into collagen precoated 96-well plates for 8 hours. The cells were fixed and immunofluorescently stained for CXCR4 and PDGFR-beta. The cell nuclei were stained with Hoechst33342. Cell images were acquired using the Cellomics ArrayScan HCS Reader (40× objective) and analyzed using the Target Activation BioApplication Software Module. (A) Fluorescence intensity of CXCR4 in non-irradiated A549 cells (brown), non-irradiated sphere cells (green), and radiation survived sphere cells (red) plotted against the object area. Each spot represents a single cell. Cells to the right of the black line are positive (above IgG control staining). (B,C) The total average fluorescence intensities of CXCR4 (B) and PDGFR-beta (C), in non-irradiated bulk NSCLC cells (grey), in the radiation survived adherent cells (green), in the non-irradiated sphere cells (red) and in the radiation survived sphere cells (blue) are shown. Fluorescence intensities of respective IgG controls were subtracted. Each point presents average intensities (pixels) estimated for 3000 cells.