Cl 100 temperature controller

Demuscled fillet preparations were made from GAL4^19-12>UAS-mCD8::GFP larvae housed in Petri dishes lined with Sylgard 184 (Dow Corning), which were filled and constantly superfused with HL-3 saline. Extracellular recordings were made with a pipette (tip diameter, 5–10 μm) connected to the headstage of a patch-clamp amplifier (Multiclamp 700A, Molecular Devices). Gentle suction was applied to draw the soma and a small portion of neurite into the pipette. The amplifier was set to voltage-clamp mode to record neuronal spikes. The output signals from the amplifier were digitized at a sampling frequency of 10 kHz using a Micro1401 A/D converter (Cambridge Electronic Design) and acquired into a laptop computer running Windows 10 with Spike2 software v. 8 (Cambridge Electronic Design). To apply a low temperature stimulation, saline was passed through an SC-20 in-line solution cooler (Warner Instruments) connected to a CL-100 temperature controller (Warner Instruments). Average spike frequency was measured during baseline room-temperature conditions (60 seconds) and during superfusion of chilled saline (60 seconds at 20, 15, or 10°C).

A Nikon SMZ 745T stereo microscope (Nikon Instruments, Melville, NY, USA) with a mounted camera (acA640-120um; Basler AG, Ahrensburg, Germany) was used for live recording of MTF contractions. Devices were placed in a 35-mm-diameter polystyrene petri dish filled with low-glucose Tyrode’s solution [1.8 mM CaCl₂, glucose (0.1 g/liter), 5 mM Hepes, 1 mM MgCl, 5.4 mM KCl, 135 mM NaCl, and 0.33 mM NaH₂PO₄, pH 7.4]. A custom 3D-printed (MakerBot, Brooklyn, NY, USA) field electrode with two platinum wires spaced 1 cm apart was connected to a field stimulator (Myopacer; IonOptix, Westwood, MA, USA). The petri dish was placed in a heating plate (Warner Instruments, Hamden, CT, USA) controlled by the feedback from a thermoprobe, consisting of a cable with a bead thermistor (Warner Instruments, Hamden, CT, USA), in the solution connected to a CL-100 Temperature Controller (Warner Instruments, Hamden, CT, USA) and liquid cooling system (Koolance, Auburn, WA, USA) to maintain the solution at 37°C during the O₂ modulation and contractile stress measurement. The precut bilayer laser-engraved MTFs were manually peeled using forceps. Then, deflection of the MTFs was recorded at 2-Hz electrical pacing with an external voltage of 15 V and a collection frame rate of 100 fps.