Facs pre sort buffer

Single-cell suspensions of tumor cells were prepared as previously described using a combination of physical dissociation and enzymatic digest (24 (link)). Red blood cell lysis was performed using ACK Lysing Buffer (Gibco, Waltham, MA, USA) per manufacturer’s recommendation. Digests were filtered through a 100µm cell strainer prior to debris removal (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat # 130-109-398). Cells were resuspended in BD FACS Pre-Sort Buffer (BD, Franklin Lakes, NJ, USA; Cat # 563503) for further fluorescently activated cell sorting (FACS) analyses, or in washed 2X in sterile PBS+0.04% non-acetylated BSA for single cell sequencing.

Before sorting, cells were washed twice in 1× PBS buffer (DPBS without calcium chloride and magnesium chloride; Sigma Aldrich, D8537) and labelled with 7-AAD (BD Pharmingen, 51-68981E) for live/dead differentiation and FITC-conjugated antibody [anti-CD47 (BD Pharmingen, 556045) for A375 and anti-CD81 (BD Pharmingen, 551108) for Jurkat]. After washing off the unbound antibodies in 1× PBS, cells were resuspended in BD FACS Pre-Sort Buffer (BD, 563503). Single cell sorting in 8-tube PCR strips was done using a BD FACSJazz Cell Sorter. A375 cells were sorted in 7 μl 1× PBS buffer and Jurkat cells in 8 μl lysis solution [100 μl 10× Lysis buffer (Takara, 635013), 5 μl RNase Inhibitor (Takara, 635013) and 700 μl water]. Following sorting, tubes were sealed and subjected to a quick spin and immediately frozen on dry ice and finally stored at −80°C until use. All sorting experiments included negative controls (no cell in a well).