Hybrid cell generation. (A) CMTMR-stained DCs and CMFDA-stained melanoma (Mel Im) cells were fused using 50% PEG. Using a filter set appropriate for both emission wavelengths, DCs were detected as red cells, melanoma cells as green cells and hybrid cells as yellow cells (100x). More tumor cells than DCs are visible in different panels due to the unequal distribution of cells. (B) Hybrid cell consisting of a dendritic cell (CMTMR staining in red) and a Mel Im melanoma cell (CMFDA staining in green) (600x). Cells were prepared and analyzed as in 1A. (C) Analysis of hybrid cells using digital confocal fluorescence microscopy. After fusion of cells with 50% PEG without prior dye staining, cells were immunostained with anti-CD36-FITC/ anti-CD11c-FITC mAbs, the melanoma-specific anti-Mel1 antibody and the Rhodamine Red secondary antibody. Nuclear counterstaining was performed using Hoechst 33342. Hybrid cells were detected as dual-fluorescent cells with two nuclei. A representative image of a hybrid cell is shown (1000x). (D) To demonstrate phagocytosis of tumor cells, melanoma cells (Mel Im) were stained with PKH2-GL, admixed to DCs and treated with PEG. Samples were then stained with anti-CD36, anti-CD1a, and Rhodamine Red secondary antibody to label DCs. Analysis was performed using a digital confocal fluorescence microscope. A representative image is shown (1000x).