Name: Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips (Video) | JoVE Journal
Uploaded: 2012-02-07T12:00:00.000+00:00
Duration: 10 min 36 s
Description: Learn planar patch-clamp electrophysiology for recording cultured neuron activity using NRCC chips. This method enables giga seal formation and medium-throughput drug screening for ion channels.

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5 minutes

The authors wish to acknowledge Alexei Bogdanov for the fabrication of patch-clamp chips at the CPFC, and Hue Tran, Ping Zhao and Matthew Shiu for assistance with assembly. Naweed Syed was supported by a Canadian Institute of Health Research (CIHR) grant. Collin Luk is the recipient of NSERC and Alberta Heritage Foundation for Medical Research (AHFMR) studentships.

1. Chip fabrication
The process described in 6 results in a 3 μm thick self-standing film, a low 1 Mohm access resistance and a smooth-surfaced funnel shaped aperture that facilitates an intimate seal with the cell 9, see Figure 2. The chips are singulated and glued in Plexiglas packages with the aperture facing a hole connecting the chip to a subterranean channel. The gluing is dispensed in a way that minimizes the shunt capacitance to a nominal 17 pF. Packages are fitted with two 1.5 mm diameter and 6 mm long glass tubes as fluidic ports (Figure 3).
<img alt="Focused ion beam cross-section, microscopy image, material structural analysis at micro-scale." src="/files/ftp_upload/3288/3288fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Scanning electron micrograph of a focused ion beam section of an NRCC patch-clamp chip shows a smooth silicon dioxide surface and funnel shape that favors intimate cell contacts, and a shallow orifice that accounts for a low access resistance.
<img alt="Focused ion beam cross-section, microscopy image, material structural analysis at micro-scale." src="/files/ftp_upload/3288/3288fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 3. A chip is glued at the bottom of the culture vial in a Plexiglas package fitted with subterranean fluidics and glass tubes.
2. Sterilization, priming and testing
The following steps should be performed in a biology safety cabinet to avoid contamination or plugging of the aperture.
<ul type="disc">
	<li>Sterilize chips in a Harrick basic plasma cleaner (www.harrickplasma.com) with a 0.1-0.3 mbar residual air pressure for 15 min at the maximum power of 18 W. Other air plasma systems may be used, with comparable power density (24 mW/cm3) and keeping the product of power density and time constant. The plasma treatment also renders the chips hydrophilic, which facilitates priming.</li>
	<li>Fit glass tubes with sterile Silastic Laboratory silicone tubing, 1mm I.D. x 2mm O.D. (Cole Parmer Cat. # 96115-08), 3inch on one side, 1 inch on the other side.</li>
	<li>Fill fluidics through the tubes with sterile filtered standard Phosphate Buffer Solution (PBS), making sure no bubble is trapped.</li>
	<li>Clip the long silicone tube while pressurizing the PBS from the short side at 1 atm. A pool of PBS seeping through the aperture might be visible in the vial. Clip the pressurized supply of the PBS and detach the short silicone tube from that supply.</li>
	<li>Fill vial with filtered PBS using a syringe.</li>
	<li>Immerse an Ag/Ag:Cl electrode in the short tube and a counter-electrode in the vial. An impedance meter is used to confirm that the access resistance is between 300kohm and 3Mohm and the shunt capacitance is between 10pF and 25pF. A chip with a lower resistance is likely to have a leak, and a higher capacitance is considered improper for use as it may not capture the fastest dynamics of the cell′s electrophysiology 6. A chip with a lower capacitance or higher resistance is considered plugged, though it could be simply that a bubble is trapped in the orifice. Some chips are re-tested after 1H and found to have the correct electrochemical impedance.</li>
	<li>Flush the fluidic channel with sterile deionized water; empty the top vial and rinse with the same, twice.</li>
	<li>Immerse the packaged chip with tubes in fresh sterile deionized water for 30min then load with other chips in a sterile container filled with sterile deionized water. This ensures that chips remain hydrophilic and protected from contamination.</li>
</ul>
3. Chips preparation in cell biology lab
The following steps are performed in a HEPA filtered laminar flow hood using aseptic techniques, all solutions used in this procedure must be filtered sterilized prior to use using a 0.22μm filter.
<ul type="disc">
	<li>Open a chip container under a HEPA filtered laminar flow hood and remove chips from container face down to avoid scooping possible floating contaminants into top vial.</li>
	<li>Rinse top of vial of chip 2x with freshly filtered sterile deionized water to remove any residual debris from the chip surface.</li>
	<li>Aspirate sterile deionized water off the top vial.</li>
	<li>Connect one end of the silastic tubing attached to the fluidic channel to a pressurized syringe containing sterile filtered deionized water. In our system, syringes filled with solution are pressurized by connection to a compressed air tank set to 20psi. To ensure sterility, the air is filtered (0.22 μm filter) prior to entry into the syringe and our system also has an on/off valve that allows us to stop or apply pressure when needed.</li>
	<li>Using a hemostat, clamp the output end of the tubing.</li>
	<li>Open the on/off valves to pressurize the solution.</li>
	<li>To rinse the subterranen fluidic of the chip with fresh sterile deionized water, unclamp the output end of the tubing.</li>
	<li>To force the water up through the aperture, clamp the output end of the tubing with a hemostat.</li>
	<li>To avoid water drainage, clamp the input and output ends of the tubing with an hemostats and close the on/off valves.</li>
	<li>Detach the input end of the tubing from the syringe.</li>
	<li>Insert glass plugs into both ends of the tubing and remove the hemostats.</li>
	<li>Fill top vial with filter steriled deionized water.</li>
	<li>Place the lid of a 35 mm sterile dish into the base of a 100 mm sterile dish</li>
	<li>Place chip on top of the 35 mm lid and cover with 100 mm dish lid.</li>
	<li>Image chips to ensure that the membrane and aperture of the chips are free of debris and/or air bubbles. In our lab we use an upright microscope and a 20x long working distance objective.</li>
	<li>If debris and/or air bubbles are present, repeatedly flush fluidic channel and top vial. If the debris and/or air bubbles cannot be removed the chip is discarded.</li>
	<li>Once the chips are imaged, return to laminar flow hood and unplug both ends of the tubing.</li>
	<li>Attach one end of the tubing to a pressurized syringe containing physiological media.</li>
	<li>Clamp the output end of the tubing with a hemostat</li>
	<li>Open pressure valve, remove the hemostat from the output end of the fluidics and flush the fluidic channel with physiological media</li>
	<li>To rinse the subterranean fluidic with physiological media, open the on/off valves and remove the hemostat from the output end of the tubing.</li>
	<li>To force the physiological media up through the aperture, clamp the output end of the tubing with a hemostat.</li>
	<li>Clamp input end of the tubing with a hemostat and close the on/off valves.</li>
	<li>Remove the input end of the tubing from the syringe and plug both ends of the tubing with glas plugs.</li>
	<li>Remove the hemostats.</li>
	<li>Remove the water from the top vial.</li>
	<li>Fill the top vial with filter sterilized physiological media.</li>
	<li>Place the chip back into the 100 mm Petri dish.</li>
	<li>Place the base of a 35 mm dish into the 100 mm dish and fill it with filter sterilized deionized water to enrich humidity.</li>
	<li>Cover the dish until it is time for plating</li>
</ul>
4. Cell plating of snail neurons
The patch-clamp chips may be suitable for a variety of preparations. We are currently testing our chips with mammalian primary cortical neurons and have obtained preliminary results with cells cultured for 14 days 7, which indicates that our sterilization protocol is adequate and that the chips are not cytotoxic in long-term cultures. For the purpose of this protocol, snail neurons were chosen because they represent a simple but well-established model to study neuronal electrophysiology 11, and it is with those cells that we have obtained the most significant results to date 10. Detailed cell isolation and culture procedures have been described previously 12, 13.
<ul type="disc">
	<li>
	 Remove the outer shell from 2-3 month old Lymnaea stagnalis with blunt forceps and anesthetize animals in Lymnaea saline containing 10% Listerine. All subsequent steps performed aseptically within a laminar airflow hood with sterilized dissection equipment and solutions at room temperature.</li>
	<li>Replace physiological media in the fluidics with the appropriate recording solutionusing an Eppendorf pipette. In the case of Lymnaea neurons the solution contains (in mM): 50 KCl, 5 MgCl2, 5 ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) and 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.4; 130 mOsm)14</li>
	<li>Pin the snails into a Sylgard dish filled with Lymnaea saline and remove entire brain as previously described 13</li>
	<li>Treat the isolated brains in defined media (DM) with trypsin enzyme (0.2% Solution, Sigma, Catalog # T-9201) for 18 minutes followed by treatment in DM and trypsin inhibitor (Sigma, Soybean, Catalog # T-9003) for 15 minutes.</li>
	<li>Pin the brains into a small Sylgard dish filled with high osmolarity defined media (HODM - DM with glucose added; 750 μL of 1M glucose solution to 20 mL DM).</li>
	<li>Using fine forceps and dissection scissors, remove the outer and inner sheath of the ganglia of interest, exposing the neurons.</li>
	<li>Using a fire polished glass pipette filled with HODM and attached to a microsyringe, apply gentle suction near the cell of interest (Left pedal dorsal 1) until the neuron detaches from the brain and is suspended in the pipette.</li>
	<li>The glass pipette is then moved and dipped into individual neurochips where gentle expulsion from the microsyringe allows the neurons to be gently pushed out of the pipette and placed on top of the individual patch holes on the chips.</li>
	<li>Allow cells to sit undisturbed for a minimum of 2h at room temperature to promote their attachment to the chip surface surrounding the aperture.</li>
</ul>
5. Electrophysiological recordings
To connect the chips to the amplifier (in our case a Multiclamp 700B amplifier, Molecular Devices, Foster City, CA, USA)
<ul type="disc">
	<li>Replace the solution in the subterranean fluidic and in the chip vial with the appropriate recording solutions. In the case of Lymnaea neurons , the upper culture chambers are filled with Lymnaea saline (in mM: 51.3 NaCl, 1.7 KCl, 4 CaCl2, and 1.5 MgCl2) buffered to pH 7.9 with HEPES.14</li>
	<li>For stability, glue the chip onto a glass slide and place it under a microscope.</li>
	<li>To connect the chip to the amplifier (in our case a Multiclamp 700B amplifier, Molecular Devices, Foster City, CA, USA), cut one end of the tubing and insert a silver wire which is connected to the head-stage.</li>
	<li>Place the reference electrode in the top vial of the chip.</li>
	<li>The chip is now ready for recording.</li>
	<li>Apply a 5 mV step with the amplifier to measure total resistance (Rt) and determine the configuration of the neurons: cell-attached (Rt &gt; 1 GΩ); whole-cell (Rt = 50-100 MΩ plus capacitive transients, indicative of membrane rupture over the aperture); or no seal (Rt &lt; 5 MΩ). In our last set of experiments 10, 58% percent of cells had high-resistance seals and, of those, 80% of cells showed excitable responses.</li>
</ul>
6. Representative Results
<ul type="disc">
	<li>Apply depolarizing current pulses (20 pA increment) to the neuron (in this case LPeD1).</li>
	<li>Hold the neuron at Vm close to its resting potential (~ - 60 mV).</li>
	<li>Observe the responses to these depolarizing pulses. Overshooting action potentials should be observed if the neuron is viable.</li>
	<li>Figure 4 shows a representative result which is discussed in details in 14, 15.</li>
</ul>
<img alt="Voltage clamp experimental results, graph of membrane potential vs time, electrophysiology data." src="/files/ftp_upload/3288/3288fig4.jpg" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. Voltage responses (top) of a LPeD1 neuron to graded series of intracellular current pulses (below). The current pulses were applied at Vm = - 60mV.
Troubleshooting
<ul type="disc">
	<li>Before plating, image chips to determine if membrane and aperture are free of debris and suitable for plating. We use an upright microscope and a 20x long working distance objective. In our last set of experiments 1, 67% percent of chips were successfully primed in that manner.</li>
	<li>If debris and/or air bubbles are present discard chip and try another one.</li>
	<li>Various surface treatments (plasma) or coatings (PDL, PEI etc.) may be tried, but success may be highly dependent upon cell type used.</li>
	<li>The composition of the fluidic ("pipette") solution in critical for the formation of the giga-seal and the whole-cell. Adjust solution, paying attention to osmolarity, pH, and ionic makeup.</li>
	<li>For longer term culture, begin with media in microfluidic channel, then switch to pipette solution prior to recording via gentle gravity fed perfusion (~0.5 ml/min).</li>
</ul>

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<li>Martina, M., Luk, C., Py, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interrogation+of+Cultured+Neurons+using+Patch-Clamp+Chips%5BTitle%5D)+AND+%22Journal+of+Neural+Engineering%22%5BJournal%5D)">Interrogation of Cultured Neurons using Patch-Clamp Chips.</a> Journal of Neural Engineering. 8, 034002(2011).</li>
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No conflicts of interest declared.

NRCC's patch-clamp chip interrogation platform is a potentially powerful tool for high information content pharmaceutical assays and to investigate in vitro models of disease. Its advantages compared to glass pipettes are a low access resistance, which is an advantage to probe large cells, and despite a somewhat larger capacitance will result in comparable dynamics for smaller cells. Spontaneous cell to aperture seals have been routinely obtained, and whole cell entry has been observed to be spontaneous 14. A clear difference between chips and the glass pipette method is the fact that the probe is part of the cell culture dish and is not manually brought in contact with the cell membrane. Culturing cells, possibly part of functional networks, results in more biologically relevant models as disease models, and a different mechanism for securing high cell to probe seals 16. However, by contrast with cell suspensions, aspiration cannot be used to position a cell on the probe. Snail neurons, as other large cells, are amenable to manual positioning on top of the probe. For smaller cells requiring longer culture times, we have obviated the need for any manipulation and keep a high probability of obtaining a seal by patterning adhesion polypeptides on top of the probes to place cells on top of the probes, and demonstrated placement of cells on the probe 17,18.
NRCC is also developing a polyimide microfluidic patch-clamp chip 19 with a capacitance comparable to that of glass pipette. The ultimate goal of that project is a multiple-probes patch-clamp chip which allows the simultaneous monitoring of the electrophysiological activity of several neurons engaged in network behavior at the resolution of individual ion channels 14. This method is a high resolution complementary method to multi-electrode arrays 20.

culturing and electrophysiology of cells on nrcc patch-clamp chips

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Microfluidic setup with amplifier; chip, cells, vial in diagram for DNA analysis." src="/files/ftp_upload/3288/3288fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1">
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

The overall goal of the following experiment is to record the electrophysiological activity of cultured cells via a planar patch clamp chip. This is achieved by sterilizing, priming and testing the patch clamp chips to allow the growth of cell culture and the subsequent monitoring of its electrophysiological activity. After testing chips can be stored until they are primed again in a cell biology lab.As a next step, neurons are extracted from limbs, staus, and cultured onto the chips. Finally, the chips are connected to a patch clamp amplifier in order to monitor the electrophysiological activity of the cells. The main advantage of this technology over the existing planner patch clamp chips is that it is suitable for use with neurons establishing culture, allowing the interrogation of synaptically communicating, communicate in neurals.This method and the approach will allow better understanding of ion channel functions of excitable cells embedded within a neuronal network under both normal and pathological conditions. The implications of this method extend to pharmacology where it can be used to perform medium throughput, high information content, drug screenings for ion channels and receptor proteins. We have chosen to demonstrate this method using snail neurons because these neurons can be easily positioned and cultured.And because these neurons will reform network in culture, they make an excellent mod studies to transformation. More importantly, these chips can also be used in other cell systems such as cardiac and cancer cells. The main challenge with using planar patch clamp chips is to obtain chips with clean apertures, plating and culturing cells.Unplugged chips would be counterproductive, and therefore all chips are tested before being Used. The most critical point of electrophysiological assessment comes once the cells have been cultured. It consists in the formation of the giga seal between the cell membrane and the cheap upper two rim.All plantar patch clamp chips produced date used isolated cell in suspension and rely on suction to draw cells to the aperture to form a giga seal. GigE formation. Using our chips occurs spontaneously.While the neuron are culture and does not rely on suction, I will demonstrate the chip sterilization and screening step. Tanya will explain the loading in a cell biology lab, Colin will illustrate cell preparation and plating. Finally, Marcia will show our recordings are taken To begin this procedure, sterilize the chips in a heric basic plasma cleaner with 0.1 to 0.3 millibars of residual air pressure for 15 minutes at the maximum power of 18 watts.The plasma treatment also renders the chip's hydrophilic, which facilitates priming. Next, fit the glass tubes with a sterile cel elastic laboratory Silicone tubing, three inches on one end and one inch on the other end. Then fill the tubes with sterile standard phosphate buffer solution, making sure that no bubble is trapped.Clip the long silicone tube while pressurizing the PBS from the short end at one atmosphere. Then release the pressure in the fluidic channel. After that, fill the vial with PBS using a syringe.Immerse a silver, silver chloride electrode in the vial and a counter electrode in the short tube. Use an impedance meter to confirm that the access resistance is between 300 kilo ohms and three mega ohms, and the shunt capacitance is between 10 pico ferriday and 25 pico ferriday. Next, flush the fluidic channel with sterile deionized water.Empty the top vial and repeat the rinsing process twice. Subsequently, immerse the package to chip with the tubes in fresh, sterile deionized water for 30 minutes. Then load other chips in a sterile container filled with sterile deionized water.This ensures that chips remain hydrophilic and protected from contamination. Open a chip container under a HEPA filtered laminar flow hood and remove the chips with the top vial facing down to avoid contamination from packaging debris floating on the water surface. To remove any residual debris from the chip surface, rinse the top vial twice with filtered sterile deionized water Connect one end of the elastic tubing attached to the fluidic channel to a pressurized syringe containing sterile filtered deionized water.In this system, the water in the syringe is pressurized by a compressed air tank set to one atmosphere to ensure sterility air is filtered prior to entering the syringe. This system is also equipped with two on-off valves that allow the control of pressure when needed. Clamp the output end of the fluidics with a hemostat.Next, open the compressed air tank valve to pressurize the solution. Then open the hemostat at the output end of the fluidics to flush the chip with fresh sterile deionized water. Clamp the output end of the fluidics with a hemostat to force the water up through the aperture.To avoid water drainage, clamp the input of the fluidics with the hemostat and close the on-off valves. Then detach the input end of the tubing from the syringe. Plug both ends of the tubing and remove the hemostats.Fill the top vial with the filtered sterile deionized water. Next, place the lid of a 35 millimeter sterile dish on the base of a 100 millimeter sterile dish. Subsequently, place a chip on top of the 35 millimeter lid and cover with a 100 millimeter dish lid.To ensure that the membrane and aperture of the chips are free of debris or air bubbles, the chips are imaged. If debris or air bubbles are present. The fluidic channel and the top vial are washed repeatedly with sterile water.If the debris or air bubbles cannot be removed, the chip is discarded. Once the chips are imaged, bring the chips back to the laminar flow hood. Unplug both ends of the tubing.Attach one end of the tubing to a pressurized syringe containing physiological media and clamp the output end of the tubing with the hemostat to rinse the subterranean fluidic with physiological media. Open the on off valves and remove the hemostat from the output end of the tubing to force the physiological media up through the aperture clamp the output end of the tubing with the hemostat clamp, the input end of the tubing with the hemostat and close the on off valves. Next, remove the input end of the tubing from the syringe and plug both ends of the tubing with glass plugs.Then remove the hemostats. Subsequently, remove water from the top vial. Fill the top vial with filtered sterile media.Place the chip back in the 100 millimeter dish. After that, place the base of the 35 millimeter dish in the 100 millimeter dish and fill it with filtered sterile deionized water to enrich humidity. Then cover the dish until the time of plating.To begin this procedure, remove the outer shell from two to three month old li sags with a pair of blunt forceps. Then anesthetize the animals in limba saline containing 10%Listerine in a laminar airflow hood. Pin the snails into a cell guard dish filled with limba saline and remove the entire brain.Next, treat the isolated brains in defined media with trypsin enzyme for 18 minutes. After that, treat them in defined media and trypsin inhibitor. For 15 minutes, pin the brains to a small cell guard dish filled with high osmolarity defined media using a pair of fine forceps and a pair of dissection scissors.Remove the outer and inner sheath of the ganglia of interest and expose the neurons. Next, attach a fire polished glass pipette filled with high osmolarity defined media to a micros syringe gently applies suction near the cell of interest until the neuron det attaches from the brain and is suspended in the pipette. Having changed the media in the fluidics of the neuro chip to an appropriate recording solution, tip the glass pipette into a culture vial and gently expose the neuron on top of the aperture of the chip.Allow the cells to sit undisturbed for a minimum of two hours at room temperature to promote their attachment to the chip surface surrounding the aperture. Replace the solution in the culture dish with the appropriate recording solution For stability. Glue the chip onto a glass slide and place it under a microscope to connect the chips to the amplifier.Cut the long end of the tubing and insert the silver wire connected to the head stage. Next place the reference electrode in the chip culture dish. The chip is now ready for recording.Here shows an example of the voltage responses of a left pedal dorsal one neuron to graded series of intracellular current pulses. After watching this video, you should have a good understanding of how to record the electrophysiological activity of cultured cells with a planar patch clamp chip. Remember that in the first step we screen chips to ensure that non-used to plate cells have a plugged up Ture biology users start from the loading step and typically still find a few block chips for every batch.This new method represents an important breakthrough in developing a cheap technology that provides for the interrogation with an unprecedented level of resolution of in vitro neural networks. This technology can lead to advanced assays for therapeutic target identification, drug development, and diagnostic assessment. Further development of this technology will provide new means for health researchers to explore novel therapies for neurological diseases or disorders.Its potential extends to cardiac cells, cancer, and other cells that have electrogenic or properties.

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Research

JoVE Journal

Neuroscience

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

This protocol describes a technique called whole-cell patch clamp electrophysiology, which is a method to study electrical properties of Mauthner neurons and other reticulospinal cells in zebrafish embryos.

Whole-Cell Patch Clamp Electrophysiology: A Method to Study Electrical Properties of Neurons

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Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

One-channel Cell-attached Patch-clamp Recording

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Learn planar patch-clamp electrophysiology for recording cultured neuron activity using NRCC chips. This method enables giga seal formation and medium-throughput drug screening for ion channels.

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

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