Perforated patch with Amphotericin B

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R.Elghaba
R.Elghaba's picture
Perforated patch with Amphotericin B

 Hi all, 
I am trying to do some perforated patch recordings using amphotericin B. It has been nearly month now and I can't have even one sucessful experiement with really stresses me out :(.
 
So I dissolve amphoerticin B ( powder from Sigma) in DMSO , sonicate them  then add it to the normal intra and I keep this in ice all the time. 

I backfill the pipette with normal saline then with intra+ amphotericin B.

I do my best to do all this in darkness as amphoericin is senstitve to light.  

I noticed that the spikes of the cells increase in the begining after forming the seal then it decrease again and usually I loose the cell.
 
Any tips that can help me ?
 
Thanks in advance ...
 
 

Olf23
Olf23's picture
 Hi, 

 Hi, 
I am using this it works very well;
Nystatin: Weight A mg (=3 to 6mg) of Nystatin Add Ax100/6 microl DMSO, sonicate!! Take 5ml internal solution; add 20 microl of DMSO nystatin solution, vortex, sonicate and keep in the dark!!
Amphotericin B: Weight A mg of Amph.B Add Ax100/6 microl DMSO, sonicate!! Take 5ml internal solution; add 10 microl of DMSO Amphotericin B solution, vortex, sonicate and keep in the dark!!

I hope that it helps....

Olf23

R.Elghaba
R.Elghaba's picture
 Hi Olf23

 Hi Olf23

Thanks a lot for your reply.

Do you know to what extend amphotericin B is light sensitive? I mean : Is it very light sentitive ?
I heard that not all batches from Sigma is working .. Is this right?
Do you sonicate in between different trials of patching ? 
Do you use plastic syring ( as I read that it stick to plastic) ?
How long it take after formation of full seal to perforate?
 
Thanks a lot for your kind help.

 

Olf23
Olf23's picture
Hi Elghaba,

Hi Elghaba,

Now I am using just Nystatin, but it is the same thing:

1.Both nystatine an amphotercin B are very light sensisitive so when I am patching I put my syring under aluminium to avoid contact with the light and I am sweetching the light off also because I am patching neurons target with GFP.

2. For Sigma I have no idea basiclly they sell good chemicals but I change my Nystatin every 3 - 4 month it is not very expensive... just to be safe...

3. Yes I am using plastic syring it does not stick if you vortex, sonicate for 5 minutes you can go until 10 minutes sonication, for me it is OK... 

4.For the sonication if the  resistance of the  pipette get higher or  if the tip is not clean I vortex and I sonicate again... if I have trouble with the perforation I redo my intra pipette sollution...

5. After formation of gega seal immediately the membrane is perforated...

I hope that the answers are clear.. if you have more question please ask.

Good luck with your experiment,

Best,

Olf23

R.Elghaba
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 Hi Olf23,

 Hi Olf23,

Thanks a lot for useful tips.

Best regards, 

r.elghaba

R.Elghaba
R.Elghaba's picture
 Hi, 

 Hi, 

So I have tried all the tips and I can get spikes which are  20 mV in height.

but spikes should be 40-60mV.

Why I don't get full perforation?

Any advice ?

Thanks ,
Rasha