Isolation and Characterization of Melanoma Cells

Melanoma cells were identified and sorted by flow cytometry as described previously (15 (link), 20 (link)). All antibody staining was performed for 20 min on ice, followed by washing with HBSS and centrifugation at 200 × g for 5 min. Cells were stained with directly conjugated antibodies against mouse CD45 (APC, eBiosciences), mouse CD31 (APC, Biolegend), mouse Ter119 (APC, eBiosciences), and human HLA-ABC (G46-2.6-FITC, BD Biosciences). Human melanoma cells were isolated as cells that were positive for HLA and negative for mouse endothelial and hematopoietic markers. Cells were washed with staining medium and resuspended in DAPI (1 μg/mL; Sigma) to eliminate dead cells from sorts and analyses. Cells were examined on an LSRFortessa cell analyzer (Becton Dickinson) or sorted on a FACS Fusion Cell Sorter (Becton Dickinson). For analysis of circulating melanoma cells, blood was collected from mice by cardiac puncture with a syringe pretreated with citrate-dextrose solution (Sigma) when subcutaneous tumors reached 2.5 cm in diameter. Red blood cells were sedimented using Ficoll (Ficoll Paque Plus, GE Healthcare). The remaining cells were washed with HBSS (Invitrogen) before antibody staining and flow cytometry.