Bessel filter lpf 8

Black lipid bilayer (BLM) experiments were performed as previously described. Briefly, BLM was formed from a 20 mg/ml lipid solution of either DOPC or DOPC:Cardiolipin (3:1) in n-decane (Aldrich). The lipid solution was painted across the 200 μm aperture of a Delrin cup (Warner Instruments, Hamden, CT) to form the planar bilayer. The recording solution contained 150 mM NaCl, (symmetric), 2 mM CaCl₂ (in cis compartment)/10 mM CaCl₂ (in trans compartment), 10 mM Tris-HCl, pH 7.4. Currents across the bilayer were recorded using Planar Lipid Bilayer Workstation (Warner Instruments). The cis compartment was connected to the head stage input and the trans compartment was held at virtual ground via a pair of matched Ag/AgCl electrodes. Signals from voltage-clamped BLM were high-pass-filtered at 2.1 kHz using an eight-pole Bessel filter LPF-8 (Warner Instruments), digitized using Data Translation Digitizer and recorded on PC using in-house software developed by Elena Pavlova.

CHO‐K1 cells (CCL‐61, ATCC) were grown, transfected, and prepared for patch‐clamp as reported earlier (Ho et al., 2018 (link)). Briefly, 0.5 μg mPanx1 and 0.25 μg mGFP (plasmids) were transfected into the cells and seeded onto glass chips with Lipofectamine 3000 (#MR206795, #TR30007, Origene and #L3000‐008, Invitrogen). One to three days later, currents were acquired and subsequently analyzed with an Axopatch200B amplifier (Axon Instruments) and Bessel filter LPF‐8 at 200 kHz (Warner Instruments), interfaced via a Digidata 1550B to a PC running the pClamp 10.6 suite of software (Axon Instruments). Symmetrical bath and pipette buffer solutions contained (in mM): 25 KCl, 125 NaCl, 1 EGTA, 1 EDTA 10 HEPES, and pH 7.4. For each patch‐clamp experiment, a glass chip with cells was transferred into the chamber with a constant flow of bath solution and placed under the inverted microscope Nikon Ti‐S.