This procedure begins with the removal of the vilin membrane from Opus Cytes In order to successfully form the giga ohm resistant seals necessary for patch clamp recordings. Once the on cell patch clamp configuration has been obtained, suction can be applied to the pipette to gain electrical access to the cytoplasm of the cell in what is known as the whole cell patch configuration. The pipette tip can then be pulled away from the plasma membrane until it snaps back in order to obtain a small patch of membrane where the ecto domains of channels are exposed to the bath solution, this is known as the outside out patch configuration Go.Hi, I'm Austin Brown from the laboratory of Dr.Miriam Goodman in the Department of Molecular and Cellular Physiology at Stanford University.
And I am Brandon Johnson, also from the Goodman Lab. In a previous video, we demonstrated how to make patch clamp pipettes and sharp electrodes for electrophysiology. Today we will present an introduction to patch clamp recording of ion channels expressed in zpa.
So sites, since its development by S Sacramento and Air in the late 1970s, patch clamp recording has become established as an essential technique for electrophysiological measurement of single or multiple ion channels in cells. So let's get started. In order for the patch clamp pipettes to access the oocyte plasma membrane, the outer vilin membrane must be removed.
To do this first, prepare two sets of number five forceps. Slightly sharpen one set of the forceps with a file. Also prepare a glass transfer pipette by trimming and fire polishing a seven inch pasture pipette next to prevent the cytes from slipping, cut a circle out of a mesh grid that contains 0.8 millimeter squares and glue it onto the bottom of a 60 millimeter Petri dish.
This dish can be reused indefinitely if kept clean between uses. Then fill the Petri dish halfway with Hyperosmotic solution and place under a dissecting microscope. Now remove one to three opus Cytes from their incubation solution with a polished pester, pipette and place into the dish.
Wait 15 seconds to two minutes for the cytes to begin to shrink. As they shrink, the vilin membrane will begin to become barely visible as a transparent layer over the cyte. Longer times make the cyte easier to peel shorter times lead to healthier cells.
For patching, select a healthy oocyte without whirls or defects for peeling with one pair of forceps, gently grasp the vilin membrane without damaging the plasma membrane with the other grasp near the same spot and gently use both pairs of forceps to tear the clear membrane apart and free of the oocyte as a single piece. Next, with the pasture pipette, carefully move the oocyte to the recording chamber. Note that the cell will be fragile after vilin removal.
Now that the OC site is prepared, we are ready to form a high resistant seal or giga seal between the pipette tip and the oocyte membrane. To do this, fill a recording pipette with saline. Use the minimum volume of solution that makes good electrical contact with the electrode wire in order to minimize pipette capacitance.
Then flick the pipette several times to allow bubbles to float. Slide the pipette onto the electrode wire and tighten the holder. Next, apply positive pressure to the filled pipette with a mouth pipette.
This pressure is critical to keep the pipette clean as it crosses the liquid interface and moves toward the cell. Now find the cyte in the chamber and focus sharply on the edge of the cell with a pipette out of the bath center. The out of focus tip above the oocyte.
This step minimizes the time that the pipette spends in the bath before seal generation, which helps keep the pipette tip clean of contaminants from the solution. Next, drop the pipette down into sharp focus next to the oocyte. Bring it into close proximity, approximately 50 microns.
Using the patch clamp software, check the resistance of the pipette. The target resistance is approximately three to six mega ohms. Then zero the current using the voltage offset with a positive pressure on the pipette.
Move the tip slowly towards the cell while monitoring the resistance as the tip begins to touch the cell. The resistance will increase when the tip is in contact with the cell. Suddenly, but gently switch from positive to negative pressure of approximately the same absolute value.
The resistance should increase and cause seal formation. Usually seal formation occurs within several seconds. Occasionally 30 to 60 seconds are required.
Once the resistance is at one giga ohm release pressure to neutral, we now have an on cell patch. This method is useful for recordings in which the normal environment of the cell, such as the cytoskeleton, is critical to ion channel function. However, neither the internal nor external solutions can be varied.
In the next step, we demonstrate how to make an outside out patch, which allows variation of external solutions in order to create an outside out topology, rupture the membrane immediately after achieving a giga seal. To do this, apply a one volt pulse for one millisecond. The resistance should drop to slightly more than the initial pipette resistance.
Then move the pipette away from the cell slowly and smoothly. You should see a section of the cell pull with the pipette. Within a few seconds, the cell will suddenly snap back and the resistance should immediately return to giga own levels.
We now have an outside out patch in this configuration, the OD domains of any included ion channels are exposed to the bath. This is necessary to apply drugs such as blockers, which may only interact with the external domains of the channel. We have just demonstrated how to perform patch clamp recordings of ion channels expressed in xenobi cytes.
When performing this procedure, it's important to note that higher resistance seals in the range of 10 gig ohms are preferable and longer lived. In our experience reliably obtaining seals of this quality depends on many factors. The most critical parameters include using quality cytes with even coloration, and without whirls or defects, peeling cytes quickly in order to minimize the time in shrinking solution, pulling new dust-free pipettes on the same day of the experiment and polishing them within an hour of the experiment, verifying by inspection that the pipettes are smooth and polished, maintaining pressure from the time pipettes enter the bath until seal formation.
Okay, that's it. Thanks for watching. Good luck with your experiments and happy patching.
