Gc100fs 10

Cancer cells were washed in 1x PBS, filtered, counted and diluted to a final concentration of 60 × 10⁶ cells/ml in 2% PVP40 (polyvinylpyrrolidone, Sigma; PVP was diluted in full DMEM). Phenol red (Sigma) was added to the mixture to better visualize the injected cells in real time.
At 2 days post fertilization (dpf) embryos were anesthetized by 1x Tricaine and laterally arranged in groups of 25–50 on a 2% agarose dish. Borosilicate glass capillaries (Harvard Apparatus, GC100FS-10) were pulled to create microinjection needles which were then opened by tweezers (≈20 µm). Cancer cell mixture was filled into the capillary and ≈100–200 cells were transplanted to the blood stream of embryos through the dorsal part of the duct of Cuvier. After transplantation, embryos were recovered and washed in E3 and incubated at 35°C/K562 or 28°C/ZMEL1 until the next morning when they were sorted according to fluorescence. All embryos with an insufficient number of transplanted cells were discarded at this point. Specifically, every embryo with zero to ≈100 engrafted cells was discarded by the same scientist to sustain consistency. Embryos, which had a significant number of cells in-correctly grafted within the yolk sac were discarded as well.

Flies were sedated on ice for 5 mins before being mounted in dental wax (Kemdent, UK). Appendages, wings and proboscis were secured into the wax with thorax being positioned at a 45° angle aiding penetration of recording electrodes. Two sharpened tungsten wires (0.2mm) were inserted into the CNS through each compound eye whereas the third was positioned into the abdomen. Stimulation was conducted with a SIU5A stimulus isolation unit (Grass Technologies, USA) controlled via a S88 stimulator (Grass Technologies, USA). Positioning of both stimulating and earth electrodes was confirmed with a pulse (50V, 0.02 ms) through observation of wing twitching (DLM stimulation) and tergotrochanteral muscle contraction (TTM stimulation). Intracellular recording electrodes (20–30 MΩ) (GC100FS-10; Harvard Apparatus, WPI, USA) were filled with 3M KCl for recording. Glass recording electrodes were inserted into DLM 45a and the TTM, identified by position of the bristles on the thorax. Recordings were performed using an Axoclamp-2A amplifier (Axon instruments, USA) controlled by pClamp 10.4 and a Digidata 1440A (Molecular Devices, USA). The responsiveness of the respective muscle (DLM and TTM) to a train of 10 stimuli at 100Hz, termed “Following Frequency” were recorded.