Trouble getting a Gigaseal

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sadhujee420
sadhujee420's picture
Trouble getting a Gigaseal

I have been trying to patch L6 skeletal muscle cells for quite a long tim now. I have only manged to get the gigaseal twice. I am using EPC 10 from HEKA.
Everytime I try to patch the cells and i get a lot of variation in the resistance readings and hence i am unable to proceed. I tried calibrating the amplifier as per the User manual and I also tried fire-polishing and coating with sylguard but nothing seems to work. I am still trying to figure out the problem.
Anyone has an idea of what could be wrong? 

Simon8
Simon8's picture
I have no experience with

I have no experience with muscle cells but maybe you should have a look here:
http://www.scientistsolutions.com/t19490-unstable+whole+cell+patch+clamp.html

I think most things suggested there might also improve sealing.

Did you try to change your pipette form and opening? One says, that higher pipette resitance makes sealing easier. The form of the tip is also important, bu tI see you are already fire polishing your pipettes.

sadhujee420
sadhujee420's picture
 I have been using a thick

 I have been using a thick walled glass pipette (O.D. 1.5mm, I.D. 1.12mm). I have not tried a thin walled pipette yet. I did look at the shape of the fire polished pipette and tried to alter the opening size to upto 2.5um. The biggest problem that I have is lack of any reference. I could not find any literatature on these cells. I am going to have a automatic micromanipulator soon because I have been working with a manual micromanipulator till now. Do you think that will reduce the noise?

Simon8
Simon8's picture
My personal preference are

My personal preference are manual micorcontrollers, but both are ok and this should not be the source of your problems. At least, if your controller is ok and free of any drift.
I experienced drifting, while the headstage warms up (specially on Axopatch Headstages, wich can get quite hot, because of the active cooling of the circuits), what causes the metal to expand and drive your pipette forth. Be sure to switch on the amplifier ~30mins before you start.
Depending on the system you use, the controller could have been damaged, so that drift or vibration is induced. Drift can easily be checked under the microscope, just remeber the place, where the pipette was and look from time to time if it has moved out of focus or to another places (be aware, that also the microscope might drift here).
So MAYBE, you rproblem will be solved with the new controllers. But this means that your old ones where broken.
Do you have another setup around where you can try it?

But as I said, on your cells I have no idea. I have no decades of experience, just a few years now. Are you sure, that the cells are fine? Do they look healthy? Maybe you can try patching younger cells?

As I said, bigger openings will make sealing harder (worst case is sucking the whole cell into the pipette). My supervisor always says, one should increase resistance (decrease opening) until one gets good seals, and then try to open the cells. If opening is impossible you have to carefully reduce the resistance again, to find a good midpoint. I think this is a good rule of thumb, although it makes things more simple then they are, of course.

sadhujee420
sadhujee420's picture
 I still have the manual

 I still have the manual micromanipulator from HEKA EPC 10 amplifier that I am currently working with. I placed an order for the new automatic micromanipulator this week. So I should get that within a few weeks.
As far as the drift is concerned, I did check for it but couldn't find any significant drift. My biggest problem has been that, whenever I am close to patching the cell, the resistance readings go heywire and that's why I am unable to decide if the gigaseal is really formed or not. I do see capacitance peaks when I am close to patching, but as soon as I increase the gain of the amplifier, it vanishes. The cells look pretty healthy. May be tomorrow I will post a picture of it so that you can also have a look and let me know.
By the way, what kind of cells are you working with and are those easy to work with. Please do let me know.
May be I can have a try on them. Thanks anyways

Simon8
Simon8's picture
I think the best to start

I think the best to start with is dissociated neurons (these are the cells I am currently working with, but I patched in acute and cultured brain slices also; so only neurons). But as I said, I am no expert, too. An image of your cells wont help me, I do no tknow these cells.
What also can destroy the seal are vibrations. Is your table properly isolated?

Can you see anything strange happen at the pipette tip? My cells like to loose ther Nucleus and then I have it on the pipette, giving high noise and fluktuations of baseling/resistance.
So how do you make your seal? Are you using syringe or mouth to suck? Usually, no ttoo much sucking should be applied. The last step into the GOhm range of seal should be established by the membrane and the pipette without any pressure. These are the best seals. The faster the better, also. Did you check the link, I postet? In there, you'll find advice for adjusting your solutions and so on. Maybe you are also driving the pipette to far into the cell?
Just describe your approach in DETAIL.

Did you patch before on different celltypes?

Why do you change the gain of the amplifier/to what value. Is it really the very same moment, when the cell is lost?

By the way: Alot can be done wrong when patching, without anyone noticing. Then, finally it will work, and you have no idea why. This happens quite often, so do not get rid of it and just try another time with fresh solutions and a fresh mind!

sadhujee420
sadhujee420's picture
 My table is isolated and I

 My table is isolated and I am sure that is not a problem. The problem may be the manual micromanipulator itself. Because whenever I try to adjust my pipette approach by hand I see a lot of vibration in the pipette. This is because the microscope and the micromanipulator are located on the same table. Plus I use a syringe to apply suction. The L6 cells that I am using usually take around 6 days to mature. So I usually do the patch-clamp on 7th day. I do not trypsinize the cells before using. I just discard the old medium and replace it with external solution, pH 7.4. I prepare the pipettes before hand and store them in an airtight container. On the day of the experiment I just take them out and fill them with the internal solution. I admit that I may be applying a little extra suction during the patch-clamp instead of just pushing the pipette against the membrane. I did check the link that you sent me. I have noted down the compositions of external and internal solutions from that post. i will try them in coming days. During the experiment, the gain is around 0.2mV/pA and as I touch the membrane of the cell I adjust the gain to around 5mV/pA. I have not tried any other cells before. I will also be getting a microscope-digital camera along with the automatic micromainpulator. I think that will eliminate my vibration problem. If you have recordings of your patch-clamp would you please post them so that I can atleast have a look at your trace and resistance readings.
Thanks Once again!!!

Simon8
Simon8's picture
I do not really record the

I do not really record the resitance readings. So I have no files for you.
I apply suction by mouth. Mostly, because I learned it that way, but I also like the idea of having a "feeling" for the seal. I also do Wholecell-Recordings, but you just want a real Patch ("Cell-Attached"), am I right?

I also prepare my pipettes right before use (4 of them at once). This is a little bit inconvinient, because i have to pull pipettes every 30mins or so, but I recongnised that the quality is not so good, at least when the pipettes are stored free of any protection. Also, todays horizontal pullers do not require much work, once a programm has been established.

Be also sure, that when changing Gain of your amplifier, that the Range is not exceeded, i.e. the clipping LED is not lit. This will introduce distortions and maybe also fluctuations in the signal. If your Amplifier is clipping, reduce the gain!

Thats all for now. You can also look through old threads here in the forum, there is usually some good info in them, even if they are old.

Good luck!

Ian Thornell
Ian Thornell's picture
I do not have expience with

I do not have expience with muscle cells, however are these purely muscle cells? I know that with intact muscle fibers they need to be treated with a collagenase. This makes the membrane of the muscle accessible to patch onto it, otherwise theres a forest of ECM between the pipette and muscle cell.

sadhujee420
sadhujee420's picture
Yes, these are L6 myotubes

Yes, these are L6 myotubes derived from Rat skeletal muscle.
However, when I am patching, I check the confluency beforehand and make sure that it's not exceeding 70%. I am not using any collagenase as you mentioned. May be I should give it a thought though. If you can share your patching procedure with me, I would be thankful.

Ian Thornell
Ian Thornell's picture
I have not actually patch

I have not actually patch onto any form of muscle. I know from early endplate potential literature that the extracellular matrix makes patching without collagenase difficult. You can try searching muscle patch clamp for protocols in google scholar.

sadhujee420
sadhujee420's picture
 I just got MP-225 motorized

 I just got MP-225 motorized micromanipulator system from Sutter Instruments. This will probably reduce the noise and help me get a stable seal; atleast I hope so. Do you think it will improve patching conditions?

sadhujee420
sadhujee420's picture
 Does trpysinizing cells

 Does trpysinizing cells before patching help?

Sudas_1
Sudas_1's picture
If you are still having

If you are still having trouble, please call me at 9545454697 so we can discuss.