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.