(left) Molecular barcoding. NanoString Molecular barcodes consist of 6 fluorescent “spots” that can be any one of 4 colors each [hence arranged in 4^6 combinations (4096), of which 800 have been commercialized], each specific color combination represents a single mRNA species to be measured. The mRNA from the sample of interest (red) hybridizes to the complementary nucleic-acid attached to an optical barcode pattern representing that specific gene. While the hybridization event occurs in solution, the final digital counting of the barcode-nucleic-acid component is performed, single molecule at-a-time, stretched-out on the surface of the nCounter cartridge (spot-pattern on black background). (right) Schematic representation of the PanCancer Immune Profiling Panel. This 770-plex assay contains: 109 genes to cell surface markers capable of quantitating 24 different tumor infiltrating immune cell types and populations, 30 genes for commonly studied CT Antigens, over 500 genes for measuring immune response with a special emphasis on checkpoint regulation/signaling, 40 PanCancer Reference Genes. The panel can be further “customized” (by individual researchers) with the addition of up to 30 genes with our Panel-Plus feature.