Anti cd34 bv421

Mice bearing skin tumors were euthanized and the tumors were collected on ice. Each tumor was cut into small pieces and incubated with 0.5% trypsin (diluted in keratinocyte serum-free medium, Gibco) on a horizontal shaker at 37°C for 1.5 hr. Using an 18G syringe, digested tumor cells were physically isolated into a single cell suspension. The trypsin was inactivated by adding chelexed FBS. After serial filtering with 70μm and 40μm strainers (BD sciences), tumor cells were centrifuged at 1200 rpm, 4°C for 10 min. Cell pellets were resuspended with PBS containing 4% chelexed FBS and then transferred into FACS tubes with a 40μm filter. The following fluorophore-conjugated antibodies were used: anti-CD34-BV421 (BD sciences, 562608, 1:50) and anti-CD49f-PE (eBiosciences, 12-0495-81, 1:200), anti-GLUT1-A647 (Abcam, ab195020, 1:100). Propidium iodide (Sigma, P4864, 1:1000) or Zombie NIR fixable viability dye (Biolegend, 423105, 1:100) were used to negatively select live cells. Proper isotype controls, single color controls, and FMO controls were used in every experiment to set up optimal compensation and gates. Cells were analyzed and sorted using a FACSAria II (BD). Obtained data were analyzed by FlowJo. An exemplary gating strategy is described in Supplementary Information Figure 1.