A mpc involves the use of a glass pipette that is manoeuvred to an adherent cell approach. Contact is then made with the cell, and through application of negative pressure, an electrical seal forms, in the order of g. Microfluidic system for planar patchclamp electrode arrays. Whole cell patch clamp recording performed on a planar glass. Denhoff 1, anne charrier 5, tanya comas 2, robert monette 2, anthony krantis 6, naweed i. Microchip amplifier for in vitro, in vivo, and automated whole cell patchclamp. Next, the key steps of a typical whole cell patch clamp experiment are outlined, in which a currentvoltage iv curve is generated.
From understanding cellular function to novel drug discovery. The patch clamp technique has been applied to many other cell types from an array of plant species table 1 assmann and romano 1999. U v causes a change of the mechanical membrane tension. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and. Dynamite 8 dynamic clamp used to model i k1 on action potentials of hipsc cardiomyocytes. However, when processed on the patchliner, trypsintreated cells demonstrated low catch rates of 54. The patch clamp technique has been applied to many other cell types from an array of plant species assmann and romano 1999. The device consists of a planar chip made from fused quartz. Whole cell currents recorded in voltage clamp mode reveal. Microfluidic system for planar patch clamp electrode arrays xiaohui li yale university 2006 the patch clamp has been widely accepted as a standard technique for fundamental studies of ion channel proteins, and discovery of drugs that affect these proteins.
Frontiers from understanding cellular function to novel. Fully automated planar patch clamp instrument recording from up to 8 cells simultaneously. Nanion portapatch planar patch clamping automate scientific. Finally, applications of patch clamp recording are provided to demonstrate how the biophysical properties of ion channels, cell excitability, and neuroactive compounds are evaluated in neurophysiology labs today. Patch clamp recordings in the whole cell configuration, and single channel recordings in the cell attached configuration, can be performed with planar patch clamp chips. A tree of events for wholecell configuration establishing gray rectangle 1 and personalized cftr recording gray rectangle 2. Planar patchclamp force microscopy on living cells. Development of planar patch clamp technology and its. Problems with the quality of voltageclamp may arise with large currents and these need to be addressed by adoption of suitable. Here we report on a planar, microstructured quartz chip for whole cell patch clamp measurements without micromanipulation or visual control. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. This aspect is exploited with the internal perfusion system for the portapatch. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets.
Robotic multiwell planar patchclamp for native and primary. Other gentler enzymes like accutase, collagenase, and dispase produced cell clumps that were unsuitable for planar patch clamp fig. These planar patch clamp systems have properties of a high throughput, the automatic formation of a high seal resistance, the involvement of voltage and current clamps, and the capability of whole cell and single channel recordings, drug perfusion and data analysis. However, many of the protocols and notes given in this chapter can be applied to other automated patchclamp platforms.
Planar patch clamp approach to characterize ionic currents. Kcurrents were identified in the neural mouse stem cells as shown in the raw data traces below. Automated electrophysiological and pharmacological evaluation. Wholecell patch clamp can be used to characterize the maturation of neuronal cultures, both at the level of individual cells and at the networks connectivity level. Wholecell patchclamp recordings for electrophysiological. Automated patch clamp meets highthroughput screening. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and. Nanions planar patch clamp chips offer the unique possibility to perfuse the intracellular side of the membrane. Using automated patch clamp electrophysiology platforms in. Frontiers development of automated patch clamp technique. Automated electrophysiological and pharmacological. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in. We have investigated multiwell planar patch clamp for studies of whole cell macroscopic currents but the reader should be aware that single channel events are detectable in planar cell attached patch clamp recordings8 and it might be possible to generate a configuration similar to the insideout patch if the whole cell membrane is permeabilised.
These planar patch clamp systems have properties of a high throughput, the automatic formation of a high seal resistance, the involvement of voltage and current clamps, and the capability of wholecell and single channel recordings, drug perfusion and data analysis. Whole cell currents of hek293 cells stably expressing hnav1. Whole cell recordings performed with the portapatch. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. Patchliner versatile and sophisticated the patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously. The herg safety service is performed at our parent company, evotec, and is a cellbased assay which employs the qpatch htx system sophion bioscience as or the syncropatch 384pe nanion technologies as automated patch clamp electrophysiology measurements. May 12, 2017 whole cell patch clamp recordings from morphologically. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole cell configuration. The portapatch is a miniaturized patch clamp system supporting gigaseal recordings from one cell at a time. Wholecell patchclamp electrophysiology of neurons is a goldstandard technique for highfidelity analysis of the biophysical mechanisms of neural computation and pathology, but it. Using automated patch clamp electrophysiology platforms in painrelated ion channel research. We have investigated multiwell planar patchclamp for studies of wholecell macroscopic currents but the reader should be aware that single channel events are detectable in planar cellattached patchclamp recordings8 and it might be possible to generate a configuration similar to the insideout patch if the wholecell membrane is permeabilised.
In combined patchclampafm measurements, different configurations between the patchclamp pipette, the afm tip and the probed cell have so far been realized fig. Whole cell current responses of hek293 cells expressing na v 1. Automated wholecell patchclamp electrophysiology of neurons. Whole cell patch clamp recordings from morphologically. The basic principle of wholecell recording is holding the membrane potential of a cell to study its function by way of recording the ionic currents. It offers fast and easy access to high quality patch clamp data with only minimal training.
Described here, is a simplified protocol of the wholecell patch clamp. The portapatch is a onecellatatime system, which is surprisingly small for a complete patch clamp rig. In this study we complement the set of possible configurations by combining the afm with a planar patchclamp chip fig. The system uses nanions planar patch clamp chips and generates high quality data. Indeed, synaptic transmission, or the relaying of signals from one neuron to another, is regulated by the opening and closing of these transmembrane proteins 1. The patchclamp technique was originally developed in the late 1970s 25 and further improved by hamill et al. Whole cell patch clamp recording performed on a planar. Automated wholecell patchclamp electrophysiology of. Nanions planar patch clamp chips offer the unique possibility to perfuse the intracellular side of. Several books have been written describing this technique in detail. Dissolve 4 mg of agar in 100 ml of 3m kcl solution by boiling and transfer to 10cm dishes.
A new technique for multiple reuse of planar patch clamp chips. Introduction he opening and closing of ion channels in cell membranes is the basis of many cell functions. Instead of positioning a pipette on an adherent cell, cell suspension is pipetted on a chip containing a microstructured aperture. Electrophysiological recordings were obtained through the use of the wholecell mode of the planar patchclamp technique portapatcho nanion, munich, germany fertig et al. Patch clamping is still a laborious process, requiring a skilled experimenter to micromanipulate a glass pipette under a microscope to record from one cell at a time. All cells were recorded in the whole cell mode of the patch clamp technique using the syncropatch 384pe incorporated into a biomek fx pipetting robot beckman coulter or the patchliner octo. Instrumentation based on the planar patch clamp principle is made available by nanion technologies gmbh. Whole cell patch clamp recording performed on a planar glass chip.
The tip of the pipette figure 1a is replaced by a micronsized aperture through a selfsupported thin film figure 1b separating an upper cell suspension chamber from an underlying pipette chamber. Suction is applied to the pipette interior to seal the cell membrane onto the tip. The combination of a patch clamp setup with an ultrasonic system overcomes this obstacle and allows the study of sonoporation. It is important to note that different cell types require modifications to the. Here we describe features of nanions npc16 patchliner and processes and protocols suited for this particularly flexible and successful highthroughput automated platform, which is based on planar patchclamp technology.
Limitations of quantitative wholecell voltage clamp posted on may 9, 2017 by p. A single cell is then positioned on the hole by suction and a tight connection gigaseal is formed. Robotic multiwell planar patchclamp for native and. Nanion s porta patch, claims to be the worlds smallest patch clamp workstation, and uses nanion s planar patch clamp chips providing data on any ion channel target. Conventional patch clamp conventional patch clamp technology is a powerful technology which has been used to study ionic channels. It is a research grade instrument allowing for whole cell as well as single channel recordings with great experimental versatility. Frontiers development of automated patch clamp technique to. Whole cell patch clamp electrophysiology of neurons is a goldstandard technique for highfidelity analysis of the biophysical mechanisms of neural computation and pathology, but it requires great.
One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the wholecell configuration. The abbreviations illustrate the voltageinduced membrane movement described in section 1. Experiments are performed on a single cell at a time, whereby whole dose response curves can be obtained from a single cell. Isolation and wholecell patch clamping of arabidopsis. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. The apertures replace the tip of glass pipettes commonly used. It is, however, easier to obtain reliable data when current amplitudes are larger e. Planar patchclamp force microscopy on living cells request pdf. Next, the key steps of a typical wholecell patch clamp experiment are outlined, in which a currentvoltage iv curve is generated. The advantage of whole cell patch clamp recording over sharp electrode technique recording is that the larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell. Nanions planar patch clamp devices the portapatch is a semiautomated device with a small footprint for the analysis of ion channels in cells and lipid bilayers. Figure 1 replacing the patch clamp pipette with a microstructured chip.
With its vast experimental freedom and gigaseal data quality, the patchliner is one of the most versatile patch clamp instruments on the market. Microfluidic system for planar patchclamp electrode arrays xiaohui li yale university 2006 the patch clamp has been widely accepted as a standard technique for fundamental studies of ion channel proteins, and discovery of drugs that affect these proteins. The portapatch is routinely provided with an axonmdc multiclamp amplifier or an epc10 amplifier from heka together with a computer and the software packages. The patch clamp technique was originally developed in the late 1970s 25 and further improved by hamill et al. The cells easily formed gigaohm seals, and patch clamp recordings were obtained from the whole cell configuration membrane holding potential 60 mv, applying 500 ms depolarizing voltage pulses. On the syncropatch 384pe, voltage protocols were constructed and data acquired using patchcontrol 384 nanion technologies. Patch clamping is still a laborious process, requiring a skilled.
Levis and rae, 1993 and is thus a very suitable substrate for planar patch clamp chips. Wholecell patchclamp recording has good signaltonoise ratio and can resolve current amplitudes of a few pico 1012 amperes pa. Analysis advanced techniques, chapter 14 planar patch clamping, which i. Not only a powerful research tool but also ideal for educational purposes and quick tests of cells and ion channels. Rupprecht before i first dived into experimental neuroscience, i imagined whole cell voltage clamp recordings to be the holy grail of precision. As neurons derived from axolnscs mature over time, the number of cells spiking increased up to 100% of the total number of neurons recorded at one month after plating figure 3a. The cell is immobilized and electrically accessed by the patchclamp hole of the planar patchclamp chip while mechanically probed by the afm tip. Planar patch clamp chips in systems like the nanion portapatch are. Development of planar patch clamp technology and its application in. Using optimized conditions, we recorded both wt and corrected f508delcftr clcurrents with automated whole cell patch clamp and compared the data to results obtained with conventional manual whole cell patch clamp. Fully automated dynamic patch clamp for the patchliner. The patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously.
Applied in cell culture, this technique provides accurate control of the. The patchliner is an automated patch clamp robot for medium thoughput with ultimate flexibility for assay design. Unlike other planar patch clamp devices we considered, the patchliner gave us full access to the electrophysiology modes we require for a broad range of experimental protocols including voltage clamp, current clamp, standard whole cell, cell attached, and perforated patch configurations combined with a facile ability to exchange the internal. The basic principle of wholecell recording is holding the membrane. The portapatch enables fast fluid exchange on the chip, and is suitable for both voltage gated and ligandgated ion channels. We employ planar microstructured borosilicate glass chips nanion technologies gmbh, munich, germany, used in a commercially available patchclamp workstation portapatch from nanion, which commonly is used for automated patchclamp recording purposes alone 16,17. B the two different protocols applied for cftr recording. Planar patch clamp is a novel method developed for high throughput electrophysiology. Planar patchclamp chips have been developed and refined over the last decade to specifically address the above limitation sigworth and klemic, 2005. Nanion portapatch applications automate scientific. The basic principle of whole cell recording is holding the membrane potential of a cell to study its function by way of recording the ionic currents. Due to its dielectric properties quartz is the almost ideal material for patch clamp pipettes rae and levis, 1992. The patch clamp goes planar the scientist magazine.
A whole cell configuration of the classical patch clamp technique. Patch clamp recordings in the whole cell configuration. Experimental procedure for automated patch clamp apc cystic fibrosis transmembrane conductance regulator cftr recording. Traditional patch clamp has a very low throughput which has been proven to be a. We utilized the novel wholecell mode of the planar patchclamp technique portapatch nanion, munich, germany394041 42 to detect wholecell currents in bon1 cells. The state of the art technology for the study of ion channels is the patch clamp technique.
The most popular whole cell variant of the technique detects the ensemble current over the entire cell membrane. Electrophysiology the branch of physiology concerned with electric phenomena in living tissues bioelectric potentials and the mechanism of. Together with the method of whole cell recording, patch clamp techniques permit the investigation of ion channel conductance and kinetic behavior. Limitations of quantitative whole cell voltage clamp posted on may 9, 2017 by p. Rupprecht before i first dived into experimental neuroscience, i imagined wholecell voltage clamp recordings to be the holy grail of precision. Nanions portapatch, claims to be the worlds smallest patchclamp workstation, and uses nanions planar patch clamp chips providing data on any ion channel target. Iv protocol blue dots for currentvoltage relationship. The latest progress in the research of taste cells electrophysiology and taste. All cells were recorded in the wholecell mode of the patch clamp technique using the syncropatch 384pe incorporated into a biomek fx pipetting robot beckman coulter or the patchliner octo. This information has led to the discovery of new classes of ion channels and their physiological role in cells too small to be amenable to the standard voltage clamp techniques and to cells that are. Does anyone use the automated patch clamp, the portapatch. One of the advantages of planar patch clamp is the possibility of perfusing the.
In addition, in planar patch clamp it is also possible to use internal agar bridges. The key point of patch clamp is to form gigaohm seal. A quartz substrate of 200 mm thickness is perforated by wet etching techniques resulting in apertures with diameters of 1 mm. The microelectrode pipette, whose aperture provides the suction that couples the instrument to the cell, is replaced by a planar electrode substrate into which a hole has been bored. I have used patchliner ordered from nanion, but the success rate is not as high. Another twostep protocol for largescale isolation of arabidopsis guard cell protoplasts has also been described leonhardt et al. Planar patch clamping, as the name implies, is a twodimensional or planar variation on traditional patch clamping. Isolation and wholecell patch clamping of arabidopsis guard. Miniaturized patch clamp system supporting gigaseal recordings from one cell at a time.
As in conventional patch clamp, agar bridges are used for the reference electrode. Jun 29, 2010 mouse somatosensory barrel cortex layer 23. Planar patch clamp article about planar patch clamp by. Patchclamp setup conventional patch clamping, now also referred to as manual patchclamp to distinguish it from the recently developed automated patchclamp described below see section vi, is accomplished by sealing the small tip of a pipette to the surface of the cell membrane in such a way that is possible to isolate a tiny. Portapatch planar patch clamping the portapatch is a onecellatatime system, which is surprisingly small for a complete patch clamp rig. Using an xyz micromanipulator and a binocular microscope, the tip diameter 12.