Oc 725c oocyte clamp

TEVC recordings were performed as previously described (77 (link)). A regular ND96 solution for TEVC contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM Hepes (pH = 7.5). Ionic currents were recorded in TEVC using an OC-725C oocyte clamp (Warner Instruments), low-pass filtered at 1 kHz, and sampled at 5 kHz. Microelectrodes were pulled using borosilicate glass to resistances from 0.3 to 0.5 megohms when filled with 3 M KCl. Voltage clamp data were digitized at 5 kHz (Axon Digidata 1440A; Molecular Devices), collected using pClamp 10 (Axon Instruments). Note that while ordinarily oocyte data using monomeric (single) constructs would better reflect the decrease in amplitudes, this is not the case with the tetrameric constructs because the synthesis of the long RNA molecules is not very efficient, and it is difficult to know how much of the complete sequence is actually being injected into each oocyte.

The open reading frames (ORFs) encoding MsepOR8, MsepOR27, and MsepOrco were amplified and cloned into the pT7Ts vector. Subsequently, cRNAs were synthesized using the mMESSAGE mMACHINE T7 kit (Ambion, Austin, TX). Electrophysiological recordings were conducted following previously reported protocols^{71 (link),72 (link)}. Mature healthy oocytes (stage V–VII) (Nasco, Salida, California) were treated with collagenase I (GIBCO, Carlsbad, CA) in washing buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl₂, and 5 mM HEPES [pH = 7.6]) for ~1 h at room temperature. After overnight incubation at 18 °C, oocytes were microinjected with 27.6 ng ORs cRNA and 27.6 ng Orco cRNA. Post-injection, oocytes were further incubated for 4–7 days at 18 °C in 1X Ringer’s solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl₂, 0.8 mM CaCl₂, and 5 mM HEPES [pH = 7.6]) supplemented with 5% dialyzed horse serum, 50 mg/ml tetracycline, 100 mg/ml streptomycin, and 550 mg/ml sodium pyruvate. Whole-cell currents were recorded from the injected Xenopus oocytes using a two-electrode voltage clamp. Odorant-induced currents were measured with an OC-725C oocyte clamp (Warner Instruments, Hamden, CT) at a holding potential of −80 mV. Data acquisition and analyses were conducted using Digidata 1440 A and pCLAMP 10.2 software (Axon Instruments Inc., Union City, CA). The tested compounds were dissolved in dimethyl sulfoxide (DMSO) to 1 M stock solutions and stored at −20 °C. Prior to testing, the stock solutions were diluted with 1 X Ringer’s buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl₂, 0.8 mM CaCl₂, and 5 mM HEPES [pH = 7.6]).