I try to patch cardiomyocytes but I only have “bad” signal.
I think the medium is OK.
The problem is the quality of the seal or an error of configuration of the software. I work with pClamp 9 and 10.
Could someone, who knows this software, help me to verify the configuration?
First try using the model cell to determine if your equipment is configured properly. Molecular devices has great tech support to help you with this end of the problem. After that, you will have to determine if your cell isolation is good.
It would be helpful if you would describe your problem in more detail. What exactly do you mean with a "bad" signal?
In general the use of a cell model with known parameters (resistance, capacity) should be the first approach to check the configuration.
May be this site helps you (attached link).
http://mdc.custhelp.com/app/answers/detail/a_id/17005/~/membrane-test-guide
One problem I can figure out from your examples is the membrane potential, which is already quiet depolarized (around -30 mV). This could be based on cell quality or quality of the seal.
You can try to apply a holding current to realize a membrane potential below -60 mV – just to see whether AP configuration changes. Generally untreated wildtype cardiomyocytes should display a resting membrane potential below -60 mV!
Generally I would recommend to determine the AP-trigger-threshold first. Create a pulse that increases current amplitude every sweep. You should see “passive” charging of the membrane first until you reach the threshold. Than use a current amplitude around 1.5x the amplitude that was sufficient to trigger an AP.
What species do you patch? Rodent cardiomyocytes?
Agree with Jan Sebastian and Martin on this one. The depolarized resting membrane potential would be a sign of a poor seal or poor quality cells..Which solutions are you using for these recordings (intra and extracellular)? Are you seeing any spontaneous activity in your preparation?
Okay, so we are talking about hIPS-cardiomyocytes.
This means that you can expect a heterogeneity regarding resting membrane potentials and AP configurations as you can see in the company’s flyer and in many publications which is typically attributed to the presence of ventricular-like, atrial-like and pacemaker-like CMs.
I do not have experience with commercial hIPS-CMs but with “custom made”. Depending on the quality of the preparation you will also find cells showing atypical APs which cannot classified into the above mentioned groups.
In general I would recommend to use intracellular/extracellular solutions from established/published protocols using this cell line (see links).
Are you sure about the 7 mM CaCl2 in your extracellular solution?
But before you start changing solutions – how do you check for seal quality? Do you work with ruptured or perforated patch? What is the geometry and resistance of your pipettes?
As you gathered from our comments seal quality is one possible problem. But you have to be able to check and estimate it.
http://axiogenesis.com/images/pdf/flyers/Axiogenesis_Cor4U_hiPSC_Flyer.pdf
http://axiogenesis.com/images/pdf/appnotes/CytoPatch_Cor4U_Cardiomyocytes-AppNote.pdf
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