Hi! Dont know how useful will be my answer but from my expirience with Amphotericin B which I used for my perforated patch experiments, I can suggest that it is very critical to have your antibiotic solution always freshly made and it is better to make new one every few hours to have it always being active.

Best,

Miroslav

Riccardo,

Does your experiment require specifically Gramicidin?

Hi Riccardo,

here my procedure:

I used to prepare a freshly made stock solution the day of the experiment (as Miroslav said, this is an important point). The procedure was: 1- weigh a small quantity of gramicidin on a high precision balance 2- dilute with appropriate volume of high quality DMSO (avoid using an old DMSO bottle in use, DMSO gets easily hydrated) to obtain a stock concentration of 60 mg/mL; 3- just before recording add the stock to your pipette solution to obtain 30 µg/ml and sonicate briefly (8-10 s twice); 4- fill the tip of your pipette with gramicidin solution, then add normal solution in the rest of the pipette (a small air bobble between the tip and the body of the pipette helps slowing down the dilution of the gramicidin in the tip).

Hope this is helpful.

Best,

Ciao, Francesco

Hi everybody and thanks a lot for your suggestions!

I have a protocol in which I have to prepare a stock solution (Gram+DMSO,  to vortex and strongly sonicate) to store at -20°C and thaw every time I do an experiment. On the day of the experiment, diluite the stock with INTRA solution, again vortex and sonicate. I know to keep GRAM in dark and ice...

A person told me to put in the pipette ONLY Gram-intra and not a bit of Gram-free in the tip, and that this way the antifungal takes less time to work!

And.. Do you confirm that capillars without filament are recommended?

The capillars I used were with filament.

Good luck with your experiments.

Hi everybody!!

Another query: I find different opinions about saving or not the gramicidin stock solution, because someone think it's better to use it freshly and made it every day, somone instead told me that the stock solution can be saved frozen at -20 °C, and thawed every day before use.

What do you advice according to your experience?

http://www.abcam.com/Gramicidin-A-ab144510.pdf

I would suggest making a fresh stock solution (which is waht I used to do): it takes few minutes of your time and you won't live with the eternal doubt: "maybe it doesn't work becasue the stock is not fresh".

Ok thank you so much, actually that's wthat I thought to do...

And how do you prepare the stock? Do you sonicate it only few seconds without vortex or centrifuge? I vortex it, sonicate for 2 minutes and vortex again. But not often it works, that's why I bother you so much!!   :(((

I prepare a 5 mg/ml stock......

Maybe your stock is a bit diluted. As I stated above, mine was 60 mg/ml in DMSO and final concentration is typically 30-60microg/mL. In that way, you introduce a very small quantity of DMSO in your pipette. I think  sonication should be shorter, just few seconds otherwise you may cause alterations in the protein. Try 10 sec twice with an interval of 30 sec/1 min between cycles.  My gramicidin was from Sigma, but I know other people prefer gramicidin A from calbiochem. 

I guess also the size and the geometry of your pipette may have a role. I used 3-4 Mohm pipette (a bit larger than those I used for whole cell). Anyway, when you find an experimental setting that works, go ahead with that, soon or later it should work again.

Finally, I've never met a patch clamper happy with gramicidin. the rate of success is pretty low and this can be quite frustrating

Hi and happy new year!

Now I can perforate quite well, with a final concentration of 80-100 microg/ml of gramicidin. Sometmes at the beginning of the experiment I can not obtain the Gigaseal, I guess because gramicidin is too strong, and becaming less efficient with time I can start to seal, but this is a bad story that makes me lose a lot of time...

Anyway my Ra diminishes quite well while perforating, but often the Rm stay high (around 1GOhm), and this makes my recordings more difficult!   :(

I know that everybody hate gramicidin, and actually I think the mayor difficulty is not to perforate, but to obtain good perforations!!!

Do I have to perfuse the solution once I get the giga seal? Because amphotericin might make holes on the whole cell and damage the seal

Hi, I have never used amphotericin, but gramicidin also damage the seal, that's why we use it to perforate :)

You have to fill the pipette tip with normal solution by suction, and backfill the pipette with amphotericin/gramicidin solution. You have to be very quick while approaching the cell, to avoid your reagent arrive at the pipette tip and start eating your membrane, this allows you to make the gigaseal. You can also reduce your  amphotericin concentration.

Please let me know if it's unclear...

 Hello everyone:

I was reviewing your comments about gramicidin because I want to do perforated patch. I prepared a gramicidin (Sigma; I found it on the lab) stock at 60 mg/ml in DMSO and final concentration is 30-60 microg/ml, I followed the preparation instructions mentioned here and also work in the shape of my pipette, I used aprox. 2.5- 3.5 Mohm pipette, I fill the tip of my pipette  with gramicidin solution, then add normal pipette solution in the rest of the pipette. So, of three attempts I did not get any whole-cell I stayed in cell attached, Someone mentioned that the success rate is pretty low, could be 2 out of 10 or 1 out of 10?. The issue is that I have doubts about whether I am correctly preparing the compound as well as whether the placement of the same is correct since in reality they are the firts attempts. 

On the other hand looking for another compounds that are used to pierce the patch I found amphotericin B or also called Fungizone, is used for culture media. This product is supplied as 250 microgram/ml but stock concentration that is recommended to use is of 50 mg/ml to final concentration of 0.5mg/ml so I´m below the ideal concentration and possibly this brings problems in the experiment.

Recommendations?

Thank you very much

I found that room temperature noticeably affects the speed of perforated patch access, at least for me. With RT "low", around 19-20 degrees, it may not work at all, and it will start working as the day progresses and room/equipment/solutions warm up to 24-25 degrees.

Hello everyone

I recently started to do a perforated patch-clamp in VC

my objective is to record for a long time electrically evoked EPSC in the CA1

to do that I'm patching in the CA1 pyramidal layer (mouse hippo acute slice) while electrically stim in the Schaffer collater near the CA2 CA3 region

my problem is the access to the cell is still low I think around 170-150 although I'm using 100µg/ml with a 5MOhm pipette. Also, the holding current is varying sometimes a lot from zero to -0.03

The EPSC is good and I can record around 50-100pA but the signal is not steady over time which is the main reason I switched from whole-cell to PP

Does anyone have an idea how to make it more stable especially the access and holding current?