Antibodies bound to cell surface antigens were also detected on live cells by flow cytometry. Adherent cells were detached using StemProH AccutaseH cell disassociation solution (Gibco) and incubated at 2610 5 cells per sample with antibody, isotype control or cell culture supernatants for 30 minutes at 4uC. Antibodies bound to cells were detected using a FITC-conjugated anti-IgG Fc-specific antibody (Jackson ImmunoResearch). The binding of tumor-specific antibodies to cells was compared to an excess of isotype control IgG 1 antibody (Jackson ImmunoResearch). Binding of Trastuzumab across melanoma cell lines and primary human melanocytes was evaluated by subtracting the mean fluorescence intensity (MFI) values of equal amounts of isotype control. Evaluations are representative of three experiments.