Problems going to whole cell in patch clamp

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Manenschijn
Manenschijn's picture
Problems going to whole cell in patch clamp

Hello.

This forum is great, I recently found it and thoroughly enjoying it. So I decided to ask something myself, as I have some problems with patching.

I´m patching a HEK cell line stably expressing TRPM8. Forming a seal is not a problem whatsoever, especially when using fire-polished pipets I get seals real fast and with a high resistance (4 GOhm or more, is that normal?) With un-polished pipets this is lower, around 2 MOhm and seals form less easily.

The real problem is after I get the seal. It is quite impossible to go to whole cell with the fire-polished pipets. With the non-polished ones it is a bit easier, but still hard. I open the cell by applying suction with the mouth. Any suggestion as to what might be going wrong here? And to make it a bit harder still, I recently switched from regular trypsin to a different type of trypsin, which leaves the cells looking very nice and making forming a  seal easier. 

Basically the problem is that sealing got a a lot easier, but opening a lot harder.  

Any suggestions are welcome and I´m sorry for the slightly incoherent story, but it´s been a long day ;)

Edit: some aditional information

The problem is not that it is impossible to open the seal, When even the slightest zap is applied I lose the seal. With gentle suction nothing happens until I just ¨kiss¨ it a little bit harder and voila, I lose the seal.

pipet resistance is generally between 3 and 4 MOhm
bath temperature is 32 degrees Celcius

lazy
lazy's picture
I believe you use EGTA

I believe you use EGTA-containing pipette solution.
What is the concentration?
Do you have also Ca inside the pipette?

Manenschijn
Manenschijn's picture
 Hi!

 Hi!

The internal solution I´m using:

CsCl 140 mM
MgCl2 0,6 mm
EGTA 1 mM
HEPES 10 mM

Thanks!

beths
beths's picture
Perhaps instead of using

Perhaps instead of using mouth suction you could use a small ( I would suggest a 1ml) syringe. This makes it much easier to control the amount of pressure you are putting on the cell. You can start by applying say 0.1ml of suction and increasing. Sometimes it is also sucessful if you apply a small amount of suction and leave it for around a minute.

I hope this is useful!

beths
beths's picture
I should also add that it is

I should also add that it is important to remove suction as soon as whole cell is achieved, other wise you risk losing the cell. Then reapply a very small amount (again about 0.1ml from a syringe) to prevent resealing.

Manenschijn
Manenschijn's picture
 Thanks you for all the

 Thanks you for all the suggestions. At the moment I went back to using unpolished pipets. They give a lower seal (around 2 GOhm), but at least I can open them and achieve a seal resistance around 700 MOhm. It seems the polished tips are almost too perfect! But I will try your suggestion of using a 1 mL syringe. Something I´ve been thinking about as well, and maybe some of you have experience with, is to polish them slightly less using a bit less heat and time. This might give me the best of both. Just a hypothesis..