problem to seal and break the membrane

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calciumcurrent
calciumcurrent's picture
problem to seal and break the membrane

Hi all,
I am patching cultured rat vascular smooth muscle cells. I found it is difficulty to get a G ohm seal, and even if it gets to G ohm seal when I try to break the membrane it droped again. I used two step to make the pipette and the pipette resistance in solution is around 3 M ohm. Please help me. Thanks!

The FFM
The FFM's picture
Check the following.

Check the following.
Tip inner diameter - probably needs to be approx 1.5µm. Typcally my patch pipettes take 4-5 cycles of a single step protocol on a sutter horizontal puller. Depending on your solutoins, it is possible to pull a 3Mohm pipette but the tip can be too big for a stable whole cell seal.
Is the tip smooth after pulling?  You may need to pull them a littlet widder and then fire-polish the tips to get a better seal.
are the osmolarities of all your solutoins correct and balanced?
Do you have positive pressure on the pipette when you approach the cell to forma giga seal?  This helps a lot.
Once you are on the cell apply a small negative voltage when you are trying to get the giga seal (-5 to -20mV).  Go up to your holding membrane voltage as soon as you geta Gohm seal.
how are you trying to go whole cell? - are you sucking using a pipette or are mouth suction?  you may be pulling too hard when yo uare trying to break in. Personally I found mouth suction gives more control.
Some amplifers have a "zap" button that allows a more gentle break in.  Get a GOhm seal and then set the zap to minimum and give it a pulse.  Gradually incresease until it breaks in.  This can be more gentle than suction alone, and is usually good is used in combination with some ver gentle suction at the same time.
Good luck!

calciumcurrent
calciumcurrent's picture
Hi TheFFM,

Hi TheFFM,
Thanks a lot for your suggestions. I use 1 ml syringe to break the membrane and I did give any positive pressure to the pipette. The extra solution is following:NaCl 107.1, CsCl 20, NaHCO3 4,
NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10, adjusted to pH 7.4. Is the pipette big in the such solution?When I check the paper, I found some person trysin the cells and then do the patch. Is it helpful to get a whole cell seal with trypsin the cell?  Thank you very much.

The FFM
The FFM's picture
Be very careful trypsinizing

Be very careful trypsinizing the cells beforeyo upatch because if the trypsin too concentrated or you incubate too long:
1) your cell membranes become really delicate and hard to patch
2) you cleave important parts of the membrane protein which you are trying to study and therfore change their normal properties
You could try a non enzymatic cell dissociation solution if you want to this out or round up your cells before you patch them instead.