Pacific blue acd45

To enrich viable immune cells (primary uveal melanoma sample) or sort viable immune and non-immune cells (cutaneous melanoma) the samples were sorted using a FACS Influx instrument (BD Biosciences) gating on viable CD45+ (immune) or viable CD45- (non-immune) cells (Extended Data Fig. 1a). First, cells were stained for viability (Zombie NIR, 1:500 in PBS; Biolegend, San Diego, Ca; #423106) for 10 min at room temperature in the dark. Thereafter, cells were washed once with sorting buffer, collected by centrifugation and surface antigens were stained for 15 minutes on ice in the dark. The primary uveal melanoma sample was stained with Pacific-Blue-aCD45 (Biolegend, #304022). The cutaneous melanoma sample was stained with the following (all Biolegened): Human TruStain FcX (#422302), Pacific-Blue-aCD45 (#304022), PE-Dazzle594-aCD3 (#300450), PE-CY7-aCD66b (#305116), APC-aCD15 (#301908). After staining, the samples were washed twice with ice-cold sorting buffer and 1.5×10³ cells per population of interest were sorted and immediately processed for scRNA-seq.

To sort viable immune and non-immune cells the cell suspension was stained with Zombie NIR viability dye (Biolegend, San Diego, CA; #423106) in PBS (1:500) for 10 minutes at room temperature in the dark. The reaction was terminated with sorting buffer and the cells were collected by centrifugation. After collection, the cells were stained with the following antibodies for 15 minutes on ice in the dark (all Biolegend): Human TruStain FcX (#422302), Pacific-Blue-aCD45 (#304022), PE-Dazzle594-aCD3 (#300450), PE-CY7-aCD66b (#305116), APC-aCD15 (#301908). After staining, cells were washed twice with sorting buffer and filtered through a 40 μm cell strainer attached to a FACS tube and sorted into ice-cold sorting buffer using a FACS Aria II (BD Biosciences). For each specimen we sorted 12–15x10³ viable cells for the following populations: 1.) CD45⁻ cells including tumor and stromal cells. 2.) CD45⁺/CD66b⁻ cells including all immune cells but CD66b⁺ granulocytes which are reported to interfere with scRNA-seq using 10x genomics. After sorting, the cells were placed on ice and processed immediately for scRNA-sequencing.