Electrophysiology Tips & Troubleshooting

• • Also check to make sure the electrode well is filled with KCl and you have placed the salt bridge. If either of these steps were skipped, you will have an invalid reading.

• Is it okay to make all the necessary solutions ahead of time so I don’t have to worry about making them during the experiment?

  • Usually, it’s best to make just the first few solutions ahead of time. That way, if the oocyte isn’t expressing well or something goes wrong early in the experiment, we won’t have to waste or store unused solutions. If you still want to prepare somewhat, you can measure out I+I for the solutions ahead of time and just add reagents when the time comes to mix the solutions.

Preparing Oocytes for Electrophysiology

• How do I choose a good oocyte?

  • First, check the dates on the oocytes. Make sure that they are neither too old nor too recently injected with RNA (this allows time for sufficient expression of the protein of interest). Look at the oocytes under the dissecting scope. Choose an oocyte that is a decent size, intact (does not appear to be disintegrating or falling apart) and has good contrast between the yellow and brown hemispheres, if possible.

• How do I place the oocyte in the chamber?

  • The goal of this process is to orient the oocyte with the yellow side up so that it can be impaled. This process requires a bit of trial and error. Use the pipette to drop the oocyte in the chamber a bit upstream of the middle well and allow the oocyte to roll with the flow of frog ringer and come to rest in the well. If the oocyte is not properly oriented, gently use the pipette to remove the oocyte from the well and try again.

• How should the oocyte be impaled?

  • Have someone experienced train you on this, as it is a more complicated part of electrophysiology experiment set up. Overall, the micropipette tips should impale the oocyte at approximately 45 degrees on either side, or about halfway up the oocyte. You should place the tips just at the surface of the membrane to dimple it, but do not use the micromanipulators to actually pierce the membrane. Instead, tap one manipulator at a time, checking the voltage for that channel. When the voltage jumps very negative, you know that you have impaled. A picture of the locations of the micropipettes on the oocyte is below for your reference.

How do I know when to start the experiment?

• • Before starting the experiment, the voltage readings should have stabilized and be fairly consistent (usually less than 10 difference in the readings) across both channels. You should also “play” a measurement (this is different from recording a measurement; when you “play” a reading, it is not saved for the experiment file) and ensure that the resulting red line has very little slope/is almost flat and is smooth. When you are confident in this baseline reading, zero the stopwatch and begin the experiment.

Running an Experiment

• What should I do if the flow stops during the experiment?

  • Try to keep an eye on the solution so that this does not occur. If you’re using a very small amount of solution and you are worried that the cup will run dry, you can tilt the cup to pool the solution at one end and take an early last measurement if necessary to avoid using up the last of the solution and stopping the flow. However, if it does occur, make a note that the flow stopped (and when during the experiment, if possible) and include this information on your experiment analysis. You may try to restart the flow, but be very gentle and slow when pushing solution into the tube, as too much force can cause the oocyte to be pushed to the side in the chamber, possibly disrupting the contact with the pipettes or severely damaging the oocyte. If the oocyte becomes severely damaged, abort the experiment.

• One of the pipettes slipped out of the oocyte during the experiment. Now what?

  • Usually, when this happens the voltage of only one of the channels drops and reads near zero while the other channel is much more negative, unlike the voltage changes that affect both channels in response to a solution change. Turn on the microscope light, check the oocyte in the chamber using the microscope, and attempt to reimpale. Reposition the pipette corresponding to the affected channel on the oocyte so that a slight dimple forms and the voltage reads slightly negative. Very gently tap one of the adjustment knobs on for the pipette. If the voltage begins to read negative (and ultimately a similar value to the other channel), you have likely successfully reimpaled the oocyte and can continue the experiment.

• How to tell, and what to do if I get a block?

  • Blocks occur during an experiment when the end of the channel 1 or channel 2 pipettes become blocked by the cellular contents of an oocyte (ooooh-site). You can tell this is happening when you notice a random (messy) drop in the line when viewing the raw data (Figure 1). To get yourself out of this mess, you will want to spam current through your electrodes. To do this, you will flip the current switch from record to clamp (like you would when doing an actual experiment) and press the black play button (pictured below #2) until you see the line straighten and return to normal. (You won't always be able to get the block out)