Borosilicate glass capillaries

Isolation of the ER-containing membrane fractions from Bcl-2-overexpressing WEHI7.2 cells and preparation of the giant unilamellar vesicles (GUVs) were carried out using OMD patch-clamp technique as described previously [30 (link)]. GUVs were prepared from the 1:5 mixtures of the ER-containing fraction with a 10:1 diphytanoylphosphatidylcholine/cholesterol lipid combination (5 mmol/L). The patch-clamp experiments were carried out using Axopatch 200B amplifier and pCLAMP 10.0 software (Molecular Devices, Union City, CA) for data acquisition and analysis. Patch pipettes were fabricated from borosilicate glass capillaries (World Precision Instruments, Inc., Sarasota, FL) on a horizontal puller (Sutter Instrument Company, Novato, CA) and had a resistance in the range of 7–10 mΩ. Prepared vesicles were immersed in a bath solution containing 150 mmol/L cesium chloride (CsCl), 10 mmol/L HEPES, 1 mmol/L MgCl₂, 2 μmol/L free CaCl₂ [0.9 mmol/L CaCl₂ + 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTÅ)], pH 7.2. Patch pipettes were filled with the same solution.

Cells were seeded, mounted, and maintained on the microscope as above and were manipulated with a glass microneedle as described previously^{120 (link)}. Micropipettes were fashioned from borosilicate glass capillaries (World Precision Instruments) on a two-stage Pul-2 pipette puller (World Precision Instruments). A Narishige MF-900 microforge was used to form the micropipette tip into a hook with a rounded end to engage the polyacrylamide hydrogels without tearing. The forged microneedle was mounted on a micro-manipulator (Leica Leitz mechanical or Narishige MHW-3) and lowered onto the gel surface approximately 20 µm away from a cell. Once engaged, the gel was pulled approximately 20 µm in a direction perpendicular to the cell’s axis of migration. Quantification of durotactic response was calculated as the angle defined by the long axis of the cell immediately prior to durotactic stretch and its long axis 75 min after stretch and was compared to the long axis angles of unstretched motile cells at 0 and 75 minutes.

Membrane currents were recorded in the whole-cell configuration of the patch-clamp techniques using the Axopatch 200B amplifier (Molecular Devices, Union City, CA). The resistance of the patch pipettes, fabricated from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL), when filled with the intracellular solution, was 2–3 megaohms for the whole-cell recordings. In the whole-cell experiments, series resistance was compensated for by ∼70%. Currents were filtered at 1 or 2 kHz and sampled at 10 kHz. For current-clamp experiments, the pipette solution contained 140 mM KCl, 1 mM EGTA, 1 mM MgCl₂, 5 mM HEPES. Osmolarity and pH were adjusted to 290 mOsm liter⁻¹ and 7.2, respectively. Bath medium used for current-clamp experiments contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 5 mM glucose. The osmolarity and pH of external buffers were adjusted to 310 mOsm liter⁻¹ and 7.4, respectively.

Defolliculated Xenopus oocytes were injected with 0.5–150 ng of cRNA using a Nanoject II (Drummond Scientific) and incubated at 18°C for 2–5 d before recording. Incubation was in ND96 complete medium consisting of (in mM) 96 NaCl, 2 KCl, 1.8 CaCl₂, 5 MgCl_2, and 5 HEPES, adjusted to pH 7.5, supplemented with 2.5 mM sodium pyruvate and 100 µg/ml each penicillin and streptomycin. The two-microelectrode whole-cell voltage-clamp recordings from oocytes were obtained in ND96 medium with 1 mM added 4,4‘-diisothiocyanatostilbene-2,2’-disulphonic acid disodium salt hydrate (DIDS) to block the endogenous chloride conductance. Currents were obtained with an Oocyte Clamp OC-725C amplifier (Warner Instrument Corp.). Recordings were low-pass filtered at 1 kHz and digitized at 10 kHz. Electrodes were made with borosilicate glass capillaries (World Precision Instruments) pulled with a Sutter Instrument Co. P-87 pipette puller and filled with 3 M KCl.

F₁F_O ATPase vesicle (SMV) recordings were made by forming a giga-ohm seal onto SMVs in intracellular solution (120 mM KCl, 8 mM NaCl, 0.5 mM EGTA, and 10 mM HEPES, at pH 7.3) using an Axopatch 200B amplifier (Axon Instruments) at room temperature (22–25 °C). Recording electrodes were pulled from borosilicate glass capillaries (World Precision Instruments) with a final resistance in the range of 80–120 MW. SMVs were visualized by phase-contrast microscopy with a Nikon or Zeiss inverted microscope. Signals were filtered at 5 kHz using the amplifier circuitry. Data were analyzed using pClamp 10.0 software (Axon Instruments). All population data were expressed as mean ± SEM. Membrane currents under different experimental conditions were assessed by measuring the peak membrane current (in pico-amperes) minus the baseline current. The baseline current was defined as a nonspecific electrode leak current. All current measurements were adjusted for the holding voltage assuming a linear current–voltage relationship: the resulting conductances are expressed in picosiemens according to the equation G = V/ΔI, where G is the conductance in picosiemens, V is the membrane holding voltage in millivolts, and ΔI is the peak membrane current in pico-amperes minus the baseline current in pico-amperes. Group data were quantified in terms of conductance.