Pc 10 electrode puller

Dye transfer experiments on HeLa cells were performed as previously described [37 (link)]. In short, HeLa cells were cultured in 35-mm dishes until confluence. Microelectrodes were pulled from borosilicate glass capillaries (WPI) using a pc-10 electrode puller (Narishige). The electrode was backfilled with 4% Lucifer Yellow (Invitrogen) dissolved in 150 mM LiCl buffered to pH 7.2. Subsequently, the electrode was introduced into one HeLa cell and the dye was allowed to diffuse for 3 min or for 5 min in a separate set of experiments. Fluorescent cells were immediately counted using an inverted TMD-300 microscope (Nikon) equipped with a 40x phase 3 dark medium objective with numerical aperture of 0.7 (Zeiss) and a Lucifer Yellow filter (Zeiss).

ITP was measured invasively by glass microelectrode impalement through the tumor surface as previously described^{64 (link),65 (link)}. Briefly, borosilicate glass capillaries without filament were purchased from World Precision Instruments (WPI, Inc., Sarasota, FL, USA; catalog no. TW150-4), and two-step heat-pulled using a Narishige PC-10 electrode puller. Microelectrode (1–2 µm tip diameter; NaCl 3 M electrolyte) that has resistances of ~1–2 MΩ was then placed onto a holder (Warner Instruments, Holliston, MA, USA) containing an Ag/AgCl wire immersed in the NaCl solution. Using the micro-positioners, the tip of the microelectrode mounted on the holder was immersed into the mouse Ringer’s solution. Tumor nodules were secured in the non-conductive measuring chamber and electrode potential offset to 0 mV prior to impalement. The tumor tissue was impaled with microelectrode for 50 µm and ITP was recorded for 1 minute in the same positions as for the current measurement. Since the tumor surface needs to be penetrated by the glass electrode tip which may change the surface potential of the original site, three repetitive measurements were done at nearby locations. Data were acquired (sampling of 100 Hz) and extracted using pClamp 10 (Molecular Devices) and treated using Excel.

To investigate how sugar perception is encoded by GRNs on the mouthparts, tip recordings (Hodgson et al., 1955 (link)) were performed on individual galeal sensilla chaetica A-type contact chemoreceptors (Figure S1A). Electrophysiological preparations were made as already described (Kessler et al., 2015 (link); Miriyala et al., 2018 (link)). For tip recordings, sensilla were stimulated using a motorized micro-manipulator (MPC-200, Sutter Instruments, USA) for 2 s with a borosilicate (Clark capillary glass 30–066, GC150TF-10) recording electrode (15 μm tip diameter, made with a Narishige PC-10 electrode puller) containing the test solution. Signals were acquired using a non-blocking pre-amplifier (TasteProbe; Syntech, Germany), amplified (AC amplifier 1800, A-M Systems, USA), digitized at 10 or 30 kHz (DT9803 Data Translation) and stored using dbWave (version 4.2014.3.22) or DataView (version 11.5). A minimum latency period of 3 min was allowed between simultaneous recordings to avoid adaptation. Recordings were made from among the first 6 most distal sensilla chaetica on the galea of bumblebees. For each sugar, n = 6 to 8 bumblebees were tested.