Celltracker red and green

In vitro angiogenesis assay (tube assay) was performed by first placing matrigel (Cat# 354230; Thermo Fisher Scientific, Waltham; 100 μL/well) in 96‐well sterile culture plates [26]. HUVEC (5 × 10⁴ cells/25 μL) cells were mixed with endothelial medium and with conditioned medium derived from DAOY/ DAOY‐R/, UW228/ UW228‐R, UW426/UW426‐R (1 : 1 ratio), placed on the matrigel, and incubated in a CO₂ incubator at 37 °C. In other experiments, HBMECs were incubated with conditioned medium from DAOY‐R cells transduced with lentivirus expressing shRNA against ETS1 (shETS1‐1) or a nonspecific sequence (shControl). After 16 h, cells were incubated with Calcein‐AM (Cat# C3100MP; Thermo Fisher Scientific, Waltham) for 30 min and rinsed with the endothelial cell culture medium. The number of tubes formed in matrigel was determined by fluorescence microscopy and image analysis/quantification as described [26]. To distinguish between colocalized MB and endothelial cells, MB cells and HBMECs cells were loaded with cell tracker red and green (Cat#s C34552 and C2925; Invitrogen, Carlsbad, CA, USA), respectively. Cells were coincubated in matrigel for 16 h followed by fluorescence microscopy and quantification of tube formation and colocalization.