35 mm culture dish

Larvae with micro-tumors detected by fluorescence microscopy were enzymatically digested at different hpi to be analyzed by flow cytometry. Larvae were first anesthetized with 0.004% tricaine, transferred to 1.5 ml vials with calcium-free Ringer's solution 116 mM NaCl, 2.9 mM KCl, 5 mM HEPES, pH 7.2 (all reagents from Sigma-Aldrich) and incubated at room temperature for 15 min. Larvae were transferred to a 35 mm culture dish (Falcon, Franking Lake, NJ, USA) with 2 ml of trypsin 0.25% EDTA 1x solution (Gibco), incubated at 37°C and dissociation was mechanically assisted by performing up and down pipetting with a 200 µl tip every 3–4 min until a single cell suspension was visualized under the microscope (∼15 min). Trypsin was neutralized using twice the volume of 5% FCS in PBS 1×. Cell suspension was centrifuged at 3250×g for 10 min. Cell pellet was re-suspended in 100 µl of FACSFlow and run on a FACS canto II cytometer. Human cells were detected at 660/20 nm with excitation of the He-Ne 630 nm laser, to be differentiated from larvae cells. Fluorescent positive cell population was gated to carry out the proliferation analysis with FlowJo^® 10.3 software (Tree Star, Inc. Ashland, Oregon, USA) to estimate the number of cell generations.

Neurons with functional terminals were obtained by mechanical dissociation as described previously (Akaike and Moorhouse, 2003 (link); Yang et al., 2011 (link); Steffensen et al., 2018 (link)). In brief, one midbrain slice was transferred to a 35-mm culture dish (Falcon, Rutherford, NJ) filled with a standard external solution. The region of the slice containing the VTA was directly visualized through an inverted microscope (Nikon, Tokyo, Japan). A fire-polished glass pipette with a 50-μm diameter tip was mounted to a custom-constructed mechanoelectrical device for cellular dissociation. Using a manipulator, the pipette was then positioned just below the liquid-tissue interface of the VTA region. Neurons close to the surface of the slice were dissociated by horizontal vibration at a frequency of 15–20 Hz with a range from 0.1 to 0.3 mm for 2 min. The slice was then removed from the solution containing the dissociated cells. Within 20 min, the isolated neurons adhered to the bottom of the dish and were ready for electrophysiological recordings. These mechanically dissociated neurons differ from neurons dissociated using enzymatic techniques, with the latter losing most, if not all, presynaptic terminals during the dissociation process, the former can, in contrast, retain functional nerve terminals following this process.