Dm il led fluo microscope

Brains (0273-Gal4>mCherry) were dissected and dissociated as described.³³ Briefly, 24 central brains from an equal number of male and female flies were individually dissected in ice-cold DPBS (Gibco, 14190–086) and immediately transferred into 1 mL toxin-supplemented Schneider’s medium (tSM: Gibco, 21720–001 + 50 mM d(-)-2- amino-5-phosphonovaleric acid, 20 mM 6,7-dinitroquinoxaline-2,3-dione and 0.1 mM 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 mL pipette tips. Dissociated brains were resuspended in 1 mL PBS + 0.01% BSA and filtered through a 10 mm CellTrix strainer (Sysmex, 04-0042-2314). Cell concentration was measured using a disposable Fuchs-Rosenthal haemocytometer (VWR, 631–1096) under a Leica DMIL LED Fluo microscope. A typical preparation from 24 brains yielded ∼600,000 cells.

Optical microscope images were acquired on a Leica DMIL Led Fluo microscope. Confocal microscope images were acquired on a Leica DMi8 inverted epifluorescence microscope using 405 nm and tuneable white light lasers and 63× (NA 1.4) objective at the Wolfson Imaging facility at the University of Bristol. The images were analysed using Fiji (ImageJ) software.

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.