Automated patch clamp

About: Automated patch clamp is a research topic. Over the lifetime, 174 publications have been published within this topic receiving 20623 citations.

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

Abstract: 1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

Ionworks HT: a new high-throughput electrophysiology measurement platform.

Abstract: 3To address the throughput restrictions of classical patch clamp electrophysiology, Essen Instruments has developed a platebased electrophysiology measurement platform. The instrument is an integrated platform that consists of computer-controlled fluid handling, recording electronics, and processing tools capable of voltage clamp whole-cell recordings from thousands of individual cells per day. To establish a recording, the system uses a planar, multiwell substrate (a PatchPlate™). The system effectively positions 1 cell into a hole separating 2 fluid compartments in each well of the substrate. Voltage control and current recordings from the cell membrane are made subsequent to gaining access to the cell interior by applying a permeabilizing agent to the intracellular side. Based on the multiwell design of the PatchPlate™, voltage clamp recordings of up to 384 individual cells can be made in minutes and are comparable to measurements made using traditional electrophysiology techniques. An integrated pipetting system allows for up to 2 additions of modulation agents. Typical throughput, measurement fidelity, stability, and comparative pharmacology of a recombinant voltage-dependent sodium channel (hNav1.3) and a voltage-gated potassium channel (hKv1.5) exogenously expressed in CHO cells are presented. The IonWorks™ HT device can be used in biophysical and pharmacological profiling of ion channels in an environment compatible with high-capacity screening. (Journal of Biomolecular Screening 2003:50-64)

Mammalian electrophysiology on a microfluidic platform.

Abstract: The recent development of automated patch clamp technology has increased the throughput of electrophysiology but at the expense of visual access to the cells being studied. To improve visualization and the control of cell position, we have developed a simple alternative patch clamp technique based on microfluidic junctions between a main chamber and lateral recording capillaries, all fabricated by micromolding of polydimethylsiloxane (PDMS). PDMS substrates eliminate the need for vibration isolation and allow direct cell visualization and manipulation using standard microscopy. Microfluidic integration allows recording capillaries to be arrayed 20 microm apart, for a total chamber volume of <0.5 nl. The geometry of the recording capillaries permits high-quality, stable, whole-cell seals despite the hydrophobicity of the PDMS surface. Using this device, we are able to demonstrate reliable whole-cell recording of mammalian cells on an inexpensive microfluidic platform. Recordings of activation of the voltage-sensitive potassium channel Kv2.1 in mammalian cells compare well with traditional pipette recordings. The results make possible the integration of whole-cell electrophysiology with easily manufactured microfluidic lab-on-a-chip devices.

Robotic multiwell planar patch-clamp for native and primary mammalian cells

Abstract: Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.