Hello Abderaouf,

I think borosilicate capillary glass (ID-0.86 and OD-1.5 mm is OK to use for patching DRG neurons. I have used same capillary tubes for DRG patching. Try to keep pipette resistance roughly near 2 Mohm (range 1.8 - 2.5 would be OK) and do fire polish the tip after you pulled the pipette.

You mentioned in the PDF about ramp temperature that totally depends upon the life time of filament burner (of the puller) and type of glass used to test. You can go even 10 points up or down also, its not a big problem. However, try to decrease the loop number small.

During patching, you will have to try to take a feel that how much initial pressure is required on the cell by patch pipette to get good seal. The use of appropriate pressure on the membrane through your patch pipette, position of your patch on the membrane, pipette tip (should be smooth and of appropriate tip size), and health of your cultured neuron will affect the patching procedure and good seal.

Best wishes!

Thank you very much Navin Kumar Ojha ,

For the moment a Micro-forge to fire polish the tip is not available in our laboratory, is there a method to produce pipettes with smooth tips without fire polishing? or is it possible to have a Gigaseal without fire polishing the pipettes?

Any tips or recommendations would be very helpful.

Thank you again !

It is possible to get giga seal without fire polishing.In this case, you will have to make a nice pulling program so that you get tip surface clean and relatively smooth. After pulling pipette, see pippet tip under microscope ( may be at 20x objectives) if it is smooth or not. For this, you will have to try several parameters to adjust puller in a way that you get desired pippet.

Hope that will work.

Good luck!

Thank you very much Navin Kumar Ojha , for the advice.. i will try adjusting the parameters for the pipette puller and see what i get.

1. I notice that you are using filmented glass. We only use that for very sharp microelectrodes and generally use the non filamented version for whole- cell recording. This is because without heat polishing, there is a lieklyhood that the filament can protrude slightly from the electrode tip. It also increaes the surface area of glass that you are trying to seal and so this makes giga seals very difficult. Fire polishing would help. But if you dont have a polisher, swap to using the non filamented version or alternatively borosilca hematocrit glass works just as well as expensive electrode glass. This is usually the first thing I check when one of my students is having sealing problems.

2. Go into the chamber with the electrode with positive pressure and only go thorough the water interface once (you can pick up dust and dirt from the surface). We use a utube pressure system so that the pressure can be monitored. We also use it to make seals and breakthrough.

3. Because of the dust problem you should also change the bathing solution regularly if you are not using a superperfusion system.

Can you tell me what internal solution are you using? also the external one and the osmolarity of both solutions? with borosilicate glass you don't need to fire polish, and your pipettes look OK to me. On my opinion, your problem is either in your solutions/osmolarity or you have a problem with your electrode holder that cannot keep the pressure...change your washers and try it again. Are you perfusing or not? I know culture media has a lot of sticky proteins that can affect your sealing, so if you are not using ACSF as external and perfusing I would recommend to wash several times your cells before putting them in the scope.

Thank you very much Euan Robert Brown

For the moment we only have filamented glass, is it possible to form a gigaseal with that? or are we obliged to use non filamented glass?

Sorry for slow reply. As I said above I would avoid using filamented glass. Its very difficult without heat polishing to avoid a protruding filament (especially at low heats). To demonstrate this to yourself pull a high resistance 10 meg or more patch pipette with it and see if you can get a giga seal ( you will not be able to go whole cell but at least it will demonstrate that you can get a giga seal woth your cells and solutions. Another trick (don't knwo if I mentioned it above) is to ensure that your internal solution is about 10% more dilute than the external solution (so if you external is say 300 mosmoles your internal should be around 270). This really helps stability and seal formation.

Thanks very much, Euan Robert Brown. We did as you advised and we were able to form the gigaseal with the internal solution 10% diluted than external..

The problem we are facing now is opening the cells, the gigaseal is forming but we are uable to go wholecell

Good! Now check that your electrode resistance. If it is high 10 Meg or more its very difficult to make whole cell. your resistance should be around 3-7 once the giga seal has formed (2G upwards). When the giga seal is stable you should apply direct negative suction.

Thanks Euan Robert Brown . our electrode resistance is around 2.8 to 3.5 mega ohm, the seal we are getting is stable and with resistance above 2 Giga, the problem is that when apply the negative pressure either the seal is lost or the cell is going completely inside the pipette. we are stuck at this point for a while now.. not being able to get a wholecell configuration.

OK then probably the electrode tip is too large. 1) Increase resistance to 5- 6 Meg 2) very gentle suction or using the zap button instead of negative suction. We use a u tube filled with water to control making the seal. Once its formed we apply direct suction with a 5 ml syringe but if you have a big pipette that may be far to much. Some use mouth suction but as we have lots of users we prefer to control it better and not share saliva! Another problem make sure that the electrode is not moving in the holder when you apply suction and is not leaky. This can be checked visually. this results in the electrode pulling back when you apply hard suction.