Xenotransplantation of Labeled Tumor Cells in Zebrafish

Cell preparation and xenotransplantation were performed as described previously [48 (link)]. Briefly, cells (NB-1 and IMR-32) were cultured to 70–80% confluence, and then harvested and labeled with CellTracker CM-DiI (Thermo Fisher Scientific, Waltham, MA, USA). To minimize cell clumping, DNase I (250 Kunitz units/mL, Sigma-Aldrich, Munich, Germany) for IMR-32 and Benzonase (E1014-25KU, Sigma-Aldrich, Munich, Germany) for NB-1 was added to the cell suspension and washed twice with 10% FCS RPMI, twice with serum-free RPMI and resuspended in serum-free RPMI to a final concentration of 1.0 × 10⁸ cell/mL. Zebrafish embryos were anesthetized with tricaine (MS-222, 3-Amino-benzoesäure-ethylester-methansulfonat, 0.02% (w/v), Sigma-Aldrich, Munich, Germany) and embedded in 1.0% of low gelling temperature agarose (Sigma-Aldrich, Munich, Germany). 150 to 250 CM-DiI-labeled tumor cells were injected into the yolk sac of each embryo using a FemtoJet express microinjector (Eppendorf, Hamburg, Germany) and glass microinjection needles (Science Products, Hofheim, Germany). Shortly (30 min) after injection, embryos were transferred to and held at 34 °C.