The tumor-killing potential of 2 patient-derived monoclonal antibodies was assessed: one tumor-specific antibody (6_2G3), and another antibody that did not recognize tumor cells (6_2D10), both derived from the same patient (Patient 6). Both antibodies were simultaneously evaluated using a three-color fluorescent live cell imaging cytotoxicity assay. A-375 cells were plated overnight at 2x10 5 cells per well on 6-well culture plates (Corning). Using a LIVE/DEADH Viability/Cytotoxicity kit (Molecular Probes, Eugene, OR) live tumor cells were labeled with 2mM of Calcein AM 30 minutes prior to cytotoxicity assays, washed in RPMI 1640 supplemented with 10% FCS and 1% penicillin streptomycin, and re-suspended in media containing 4 mM Ethidium homodimer-1. Ethidium homodimer-1 incorporates into the DNA of dead cells and served as a label for cell death in this assay. U-937 monocytic cells expressing Fcc receptors were used as immune effector cells at a ratio of 3:1 (effectors: tumor cells) [40] . U-937 monocytes were incubated with the 6_2G3 or 6_2D10 antibody for 30 minutes, stained with the CellTracker TM Blue dye (4-chloromethyl-7hydroxycoumarin) (Molecular Probes), washed and added to the Calcein AM-labelled tumor cell cultures containing Ethidium homodimer-1. Samples were incubated and images were captured every 5 minutes for two hours in a humidified temperature controlled chamber using a Zeiss Axiovert microscope equipped with a LD-Plan-Neofluar 20x/0.4 Korr/Ph2 objective and AxioVision software system (Carl Zeiss, Jena, Germany). Following incubation, fluorescent intensities of Calcein AM-positive live tumor cells, as well as incorporation of Ethidium homodimer-1 into cells were measured and cell death was assessed with NIS-Elements BR 3 software (Nikon). The movement of effector cells in the cultures was tracked and analyzed using IMARIS software (Bitplane, Zurich, Switzerland).