Dissociation of Drosophila Central Brains

The brain dissociation protocol was adapted from previously described methods (Harzer et al., 2013 (link); Nagoshi et al., 2010 (link)). For each day of experiments, 80–100 central brains were individually dissected in ice-cold calcium- and magnesium-free DPBS (Gibco, 14190–086) and immediately transferred into 1 mL toxin-supplemented Schneider’s medium (tSM: Gibco, 21720–001 + 50 µM d(−)−2-amino-5-phosphonovaleric acid, 20 µM 6,7-dinitroquinoxaline-2,3-dione and 0.1 µM tetrodotoxin) on ice. Brains were washed once with 1 mL tSM and incubated in tSM containing 1.11 mg/mL papain (Sigma, P4762) and 1.11 mg/mL collagenase I (Sigma, C2674). Brains were washed once more with tSM and subsequently triturated with flame-rounded 200 µL pipette tips. Dissociated brains were resuspended into 1 mL PBS + 0.01% BSA and filtered through a 10 µm CellTrix strainer (Sysmex, 04-0042-2314). Cell concentration was measured using a disposable Fuchs-Rosenthal hemocytometer (VWR, 631–1096) under a Leica DMIL LED Fluo microscope, that also allowed detecting mCherry fluorescence in dissociated KCs. Cells were diluted in PBS + 0.01% BSA up to a concentration of 200 cells/µL. Thus a typical preparation from 80 brains yielded ~2’000’000 single-cells in a volume of 10 mL.