Hi,

You have to adjust the pipette pulling and fire polishing parameters. You have to attain the 1.5 Mohm pipette resistance just by adjusting the pipette pulling parameters (i.e.. heat and velocity) and fire polishing has to be done very little, though it is necessary otherwise getting a seal would be a problem. However, if you fire polish the pipettes more, then break-in will be difficult. 

Hi, in our lab we only patch rat myocytes, so I cannot do any comparisons, but what works for us is:

1) isolation optimisation. If the cells do not seal well and they are hard to break, it usually means they need to be digested a little bit more.

2) we never used zap method. For us, the approach that works well is the suction by mouth (your mouth is much more sensitive than hand so you can be more precise than with syringe). Also, we use a system that allows regulation of suction strength - basically the suction tubing is connected by a t-connector to the patch-pipette on the one arm and a manometer on the other side - during seal, this manometer (just 2 glass 5 ml pipettes connected by a tube and filled with colored water in the bottom) is open on the other side which makes the suction quite gentle. Then when we want to break the membrane, we first close the other side of the manometer, which makes the suction still gentler than direct, and only if the membrane does not break, we close the other arm of the tubing completely so that the suction of mouth is directly translated to the suction at the pipette.

3) Allowing the cell to settle in the giga-seal. This was actually introduced by our electrical engineer student - he started measuring the membrane potential of the patch - and even after the giga-seal is established, the patch voltage is not reflecting the real membrane voltage of the cell, which means that the membrane is still not completely sticking to the pipette. So, basically we only apply gentle suction to the cell at the beginning of seal formation - in well digested healthy cell the seal then forms without any suction applied. So when we get giga-seal, we check the membrane potential, and only after it reaches ~-45 mV (this may depend on the solutions you are using, so maybe wait for lower values still) we proceed to the membrane breaking.

We use 1.4-1.8 mOhm pipettes, anything less will not seal well. If you are not concerned much about the diffusion of internal solution and the compensation of serial resistance, you can use higher pipette resistance. Make sure though that the resistance is not due to the length of the pipette tip. We do not fire polish our pipettes at all in our lab.

Thank you very much for your answers! It is helpful.  I am going to check everything again. Just one detail. When I tried to keep the rat cardiomyocytes on a giga-seal aroun 1-2 minutes ( as I did before with mouse CMs) they started to contract and I lost seal pretty easy. Do you know how to avoid this?

What voltage are you applying during the gigaseal? We keep -50 holding with 5 mV stimulation.

Dear Luliia,

  I work from many years ago with rat cardiac myocytes (and sporadically with mouse CM). From your comments I think that your main problem is that myocytes are low digested. Try to increase the 1 minute the time with collagenase.

Good luck,

  Oscar

Alexandra Zahradnikova: What voltage are you applying during the gigaseal? We keep -50 holding with 5 mV stimulation.

I use 5 mV pulse and holding is 0 mV. Recently I started to use -20 mV to establish a good seal. Usually I play with holding trying to get a seal. I also try to play with a glass capillaries. And I found that  thick glass works better but I need to keep Ra  very low and sometimes it is harder to break-in.

Thank you for your help!

 To Oscar Casis: Thank you very much for replying. Yeah the main thing is to get  good quality cardiomyocytes. But cell isolation is vary time -to-time. And Unfortunately I need to work with cell which I have got. So trying to get it better! :))