Extended Data Figure 1. Representative flow cytometry gates for the isolation of melanoma cells from the blood and lymph and representative bioluminescence images of visceral organs to quantitate metastatic disease burden. Related to Table 1, Figures 1, 2 and 3.

a-d, Flow cytometry plots showing the gating strategies used to identify human melanoma cells in the blood (a) or lymph (b) of NSG mice or mouse melanoma cells in the blood (c) or lymph (d) of C57BL mice. In all cases, cells were gated on forward scatter area versus side height (FSC-H vs. FSC-A) to exclude red blood cells and cell clumps. Mouse hematopoietic and endothelial cells were excluded by gating out cells that stained positively for anti-mouse CD45, CD31, or Ter119. Human melanoma cells were selected by including cells that stained positively for HLA-ABC and mouse melanoma cells were selected by including cells that stained positively for CD146. Melanoma cells were also identified in these studies based on DsRed, which was stably expressed in all melanomas along with luciferase. e-g, Representative bioluminescence imaging of visceral organs dissected from a negative control mouse (e), and mice transplanted with luciferase-expressing human (f) or mouse (g) melanomas. (h) Evan’s blue dye was injected into a subcutaneous melanoma to expose the tumor-draining blood (white arrow) and lymphatic (black arrow) vessels. (i) Inefficiently metastasizing human melanomas were transplanted subcutaneously into NSG mice and the number of melanoma cells per microliter of blood and tumor-draining lymph were determined (n=4 or 5 mice per melanoma from two independent experiments). Statistical significance was assessed using a Kruskal-Wallis test (*p<0.05, ***p<0.001). Exact p-values are in source data files.