Cleaning#
- Using a large pipette tip, carefully blow or suction (applying positive pressure to blow away debris can be useful for initial cleaning of a larger area while suctioning off debris tends to work better for more targeted/careful cleaning of a specific target) off any cells or debris occluding your cell of interest
- It can be helpful to swipe the cleaning pipette either to the side or up while suctioning in order to pull away unwanted tissue
- If your cell is “clean”, you should see it move freely, and/or “dimple” when positive pressure is applied
Patching#
Set up the amplifier
- In V-CLAMP, dial in the holding voltage to roughly -50 mV
- Return the command switch to “OFF” (not + or -)
- Note: steps 1 & 2 are optional, you can also dial in the holding voltage to your desired voltage once you’ve formed a seal but still prior to breaking into the cell
- Turn SEAL TEST on
Fill a patch electrode with internal, making sure there are no debris or bubbles in the tip
Load the electrode
- Lock in positive pressure with the stopcock before touching the electrode to the bath
- Lower the electrode into the bath and zero the PIPETTE OFFSET
- Note: it can take time for the junction potential to stabilize and so you may have to adjust the pipette offset more than once. If it refuses to stabilize this may indicate an issue with your ground wire/electrode, often that you need to re-chloride it.
- Cancel the FAST PIPETTE CAPACITANCE by adjusting both the magnitude and time constant as needed
- Check that the pipette resistance is within an acceptable range (8 - 12 MOhms)
Slowly lower the pipette to your cell of interest and switch to a higher objective
- A micromanipulator speed of 1-3 is recommended depending on your comfort with controlling the pipette
Once you get near your cell, a micromanipulator speed of 5 is recommended
At this point you can either maintain positive pressure with the stopcock or switch to mouth pressure depending on which strategy you prefer.
- Note: If using the stopcock method and your pipette has been in the bath for a long time it can be useful to occasionally ’top off’ the positive pressure in your line.
Approach the cell
- Position the pipette tip over or to the side of the cell
- Slowly lower the tip onto the plane of the cell if coming from above or move it towards the edge of the soma if coming from the side until it forms a dimple from positive pressure
- if no dimple appears even when extremely close to the cell it may be due to insufficient positive pressure in the pipette/a blockage or insufficient cleaning of the soma
- Release positive pressure, which should cause the cell to rebound against the pipette
- If a seal has formed, you should see the seal test pulse collapse from a square wave to just intermittent capacitive transients
- Cancel the FAST PIPETTE CAPACITANCE by adjusting both the magnitude and time constant as needed
- Note: do not over-compensate the capacitance such that the transient becomes reversed in polarity, this can damage the integrity of your recording
- Note: You may need to play slightly with the slow pipette capacitance to attain optimal cancellation. Should be to a much lesser degree to the fast pipette capacitance .
- Turn on the voltage command by flipping the switch from “OFF” to (-)
- Note: it’s recommended here to explicitly measure the plateau value (deviation from baseline between the two capacitance peaks) using your acquisition software in order to calculate your seal resistance.
- Note: Again alternatively can just dial in the desired holding potential rather than having it preset. The value of the holding potential you apply may also depend on your cell type of interest but -40 to -50mV is a good ballpark.
- Break into the cell
- “Pop” into the cell with small, snappy suctions that slowly increase in intensity
- Keep an eye on the capacitive transients, in a stubborn cell you can sometimes see them subtlety increase in size even before you fully break in, this indicates you’re very close to breaking in and to avoid being too aggressive with your negative pressure.
- You will know that you have broken in once the capacitive transients dramatically come back
- (#) the baseline should stay the same, if it jumps or falls you likely lost the patch
- (##) ratio of peak-to-plateau should be 8:1 or more
- Note: This can also depend on your cell type.
- “Pop” into the cell with small, snappy suctions that slowly increase in intensity
Optional: acquire a trace for your records - the ratio of peak-to-plateau will tell you how good your access to the cell is for this recording
- Note: it’s recommended here to explicitly measure the peak-to-plateau using your acquisition software in order to calculate your input & access resistances.
Turn SEAL TEST off and switch to I=0 MODE
- Optional: acquire another trace - this will give you a good sense of the overall quality of your recording. Ideally, your cell will be sitting at a reasonably hyperpolarized potential (roughly -35 to -55 is good for most cells) and it’s spontaneous behavior shouldn’t look too crazy. If it looks a bit off in I=0, don’t panic, hyperpolarizing current might fix things
- Note: By crazy we mean in most cells, aside from action potentials, you shouldn’t see ridiculously large magnitude depolarizing and hyperpolarizing deviations from the baseline Vm value, in other words the cell’s Vm should be fairly stable. This can vary between cell types though and often through experience you will develop a better sense of what the activity of a healthy cell in your targeted population should look like.
- Optional: acquire another trace - this will give you a good sense of the overall quality of your recording. Ideally, your cell will be sitting at a reasonably hyperpolarized potential (roughly -35 to -55 is good for most cells) and it’s spontaneous behavior shouldn’t look too crazy. If it looks a bit off in I=0, don’t panic, hyperpolarizing current might fix things
Turn the HOLDING COMMAND knob to 0 in order to remove all the voltage that you dialed in previously
- If you do not do this, your cell will likely die as you switch to I-CLAMP and blast it with way more current than it wants
Switch to I-CLAMP NORMAL MODE
- Dial in a bit of (-) holding current to help the cell sit at a reasonable potential (varies by cell type but a good ballpark range to start with is around -40 to -50 mV). Most cells require at least a few pA of (-) current
- Optional: acquire a trace for your records
Begin experiments
During Experiments#
- Regularly check the input & access resistances of the cell by injecting a short (5-10 second) hyperpolarizing (-10 pA) current pulse. This will ensure that your patch quality does not degrade too much over the course of the experiment.
- Roughly every 5-10 minutes or between trials
- Note: depending on your experiment this may not be desirable but at the very least it’s recommended to measure your input & access resistances before and after each experimental recording. With experience you can also often roughly judge the health of your recording based on the stability of the measured Vm value displayed live on the amplifier, as well as by monitoring the activity of your cell on an oscilloscope. This is useful for knowing when to end a recording during long experiments but doesn’t replace explicitly calculating the input & access resistances.
Here is a guide written by Allison Baker:
📎 Patching guide (Allison Baker).pdf
Here is a recent overview of modern patch techniques. Look out for product bias and differences from the lab-standard hardware!