Name: جعل التصحيح ، وماصات أقطاب شارب مع بولير برمجة (فيديو) | JoVE Journal
Uploaded: 2008-10-08T00:00:00.000+00:00
Duration: 5 min 30 s
Description: تعلم إعداد أقطاب التصحيح والأقطاب الحادة للفيزيولوجيا الكهربائية باستخدام الساحب القابل للبرمجة. يوضح هذا الفيديو تقنيات طلاء الشمع وتلميع النار لضمان جودة الأقطاب الزجاجية.

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We thank the following funding agencies and foundations for support: National Institutes of Health, National Science Foundation, American Heart Association, Muscular Dystrophy Association, the Donald B. and Delia E. Baxter Foundation, the Klingenstein Fund and the McKnight Endowment for Neuroscience.

Pulling pipettes
Using a microelectrode puller such as the Sutter P-97 Flaming/Brown, pull a set of approximately 10-20 pipettes.
<ol>
<li>Select your capillary glass. We use borosilicate capillaries (Sutter BF150-110-10, 1.5mm outer diameter, 1.1mm inner diameter, 10cm long). Store the glass carefully so it remains clean and dust free. </li>
<li>Design a pulling program. We use a 5 step program, with descending heat and velocity at each step, and a small pull on the final step. Sutter's pipette cookbook is an excellent reference for developing suitable programs. </li>
<li>Examine the pipette tips under a microscope to determine opening diameter and smoothness. Discard rough, uneven, or irregular tips. A good pulling protocol should ensure these are rare. For standard patch-clamp recording, tip openings should be 1-3 microns in diameter. A steep taper (blunt tip) leads to lower resistance for the same opening diameter. Shape and size can be modified by pressure polishing. </li>
</ol>
Fire polishing pipettes
<ol>
<li>Set up a polishing rig (microforge) with a platinum heating filament controlled by a foot pedal. A useful kit is available from ALA Scientific (CPM-2) or a similar apparatus can be assembled piecemeal.</li>
<li>Optional: Coat the pipette with an insulator to decrease capacitance and improve noise characteristics. We use dental wax. Sylgard 184&trade; (poly-dimethyl siloxane elastomer also known as PDMS) is also an option.</li>
<li>If using wax, keep a small molten supply nearby. With air pressure on the back of the pipette to keep wax from entering the pipette, dip the tip briefly into the liquid wax and remove. With Sylgard, store prepared elastomer in frozen aliquots and paint the pipette tip under a microscope. Heat to cure the elastomer.</li>
<li>Place the pipette (coated or not) in the polishing apparatus and bring the tip ~50 microns from the filament. Keep in mind that the filament will expand when heated.</li>
<li>Recommended: Follow the separate protocol for "pressure polishing" to change the shape of the pipette for optimal resistance and tip diameter.</li>
<li>A brief heat pulse (1-2 seconds) is sufficient to remove wax from the tip of the pipette and smooth the glass.</li>
<li>Place the finished pipette in a closed box to protect from dust, repeat for 10 successful pipettes.</li>
</ol>

<ol><li>Sutter Instrument, P-97 Pipette Cookbook. , Sutter Instrument. 
 
 <a target="_blank" href="http://www.sutter.com/contact/faqs/pipette_cookbook.pdf">http://www.sutter.com/contact/faqs/pipette_cookbook.pdf</a> (2008).</li>
</ol>

The protocol illustrated here is in daily use in electrophysiology laboratories and is also used to make injection needles for cells and animals. With a programmable puller, it is easy to make pipettes for a variety of uses.&nbsp; With attention and care, the filament on your puller will last for one year or more.&nbsp; Good luck with your experiments.

<table><tbody><tr><td>Micropipette Puller</td><td>Instrument</td><td>Sutter Instrument Co.</td><td>P-97</td><td>Or similar instrument (e.g. Sutter P-87 or P-2000)</td></tr><tr><td>Glass Capillaries</td><td>Reagent</td><td>Sutter Instrument Co.</td><td>BF150-86-10</td><td>Or, similar capillary glass. To make filling the pipette easier, use a capillary with a glass filament.</td></tr></tbody></table>

making patch-pipettes and sharp electrodes with a programmable puller

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Glass capillary pipettes have become ubiquitous in laboratories for a wide variety of techniques, such as single cell electroporation, DNA microinjection and patch clamp electrophysiology To prepare glass capillary patch clamp electrodes for recordings or silicate glass is pulled on a programmable polar. The electrode tips are coated with wax, and then the tips are fire polished and stored for future use. Hi, I'm Miriam Goodman from the Department of Molecular and Cellular Physiology at Stanford University.In this video, I'll demonstrate how to make patch clamp pipettes and sharp electrodes for electrophysiology. Glass micro electrodes, also called pipettes, have been a workhorse of electrophysiology for decades. Today, these pipettes are made from glass capillaries using a programmable pull.So let's get started. To make patch clamp pipettes, we use Sutter boar silk at capillaries that are clean and dust free, and we use the Sutter P nine seven flaming ground micro electrode puller. Sutter's Pipette Cookbook is an excellent reference for developing suitable pulling programs.To begin, lift the dust cover on the puller. Then take one glass capillary tube from the box. Make sure to re

making-patch-pipettes-and-sharp-electrodes-with-a-programmable-puller

Research

JoVE Journal

Biology

This is a guide to modifying the shape of glass micropipettes.  Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance.  This is critical to obtain low noise recordings of small cells but is useful in many applications.

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment.   Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Preparation of Artificial Bilayers for Electrophysiology Experiments

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

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron’s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

A Computer-assisted Multi-electrode Patch-clamp System

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices.  Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels.  Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

This protocol details a method for directly measuring ion channel activity on intracellular vesicles using a manual endolysosomal patch-clamp system. We illustrate the method that involves enlarging endolysosomes and manually isolating these vesicles. This approach ensures that researchers can accurately replicate and apply the procedure.

Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis

تعلم إعداد أقطاب التصحيح والأقطاب الحادة للفيزيولوجيا الكهربائية باستخدام الساحب القابل للبرمجة. يوضح هذا الفيديو تقنيات طلاء الشمع وتلميع النار لضمان جودة الأقطاب الزجاجية.

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

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