Current clamp with HEKA EPC 10 and Patchmaster

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c.hill
c.hill's picture
Current clamp with HEKA EPC 10 and Patchmaster

Hi,

I'm doing current clamp using the HEKA EPC 10 amp and Patchmaster software. I have inherited the setup files and protocols from a previous student. I'm quite new to all of this and thoroughly confused by the compensation and bridge balance procedures.

1. Upon obtaining a Gohm seal in ON CELL mode I hyperpolarise the membrane (-70 mV), compensate C-fast (using the Auto button) and then I go to WHOLE CELL mode and break in.

2. I understand that I then have to compensate C-slow. The setup file macro has already set C-slow to 50 pF and 20 Mohm. If I use the Auto button for this I get something like 3 pF and 120 Mohm. The cells I'm patching are hippocampal pyramidal cells from acute slices, so are these values typical? Should I use the auto comp or keep the macro values?

3. Next is the Rs-comp. My protocol reads: 'compensate for membrane voltage errors between pipette and cell interior using the Rs-comp button'. If I remember rightly, my options here are 2 us, 10 us and 100 us - and then I have to enter a percentage. The HEKA manual reads: 'turn up the %-comp control to provide the desired degree of compensation'. How do I know when the compensation is correct?

4. The next step is to go to C-CLAMP. The HEKA software then disables Rs-comp and it becomes 'Bridge'. The macro has this set to 10 us and the manual highly recommends that 100% compensation is used.

Steps 1. and 4. I am fine with, it's just 2. and 3. that I'm really unsure about. Do I keep the C-slow values as defined by the macro or change them? How do I sort out the Rs-comp? Any help is massively appreciated, thank you.

Olf23
Olf23's picture
 Hi c.hill, 

 Hi c.hill, 

I am using the same amp and software. I am also quite new to all.  I am doing voltage and current clamp. I have also trouble, I have just small current with an ugly pic at the begening  in the IV curve and the amplitude of spike is small 40 mV.  The R series should be as low as possible if the R series is higher it not good.  

You can ask heka support jusy send them an email explain your problem they are very helpful.

Olf23