Hi everyone
we are currently working with H9c2 cell lines. We have some patching problems with them. to tackle this matter I have used higher resistence pipet( 3-6 Mohm) and working with different passages but still low eficient results obtained.(about 1 of 25 cells only patched). we eventually want to measure action potential and total K+ currents. then, do you have any advices?
We previously worked with H9c2 cells with relatively thick pipettes. Recently I am working A7r5 cells with Cm of ~ 100 pF. Now I use pipettes of 20-30 M-ohm. Giga-ohm seal is easy. Then apply modest ZAP followed by gentle suck. It works.
to Rikuo: 20 to 30 M-ohms? are you sure? what about access resistance...ect. Some K+-currents activate with fast kinetics too. I don't think you can clamp the voltage rigorously with such pipettes.
Yusuf? 1- Do you polish you pipettes? if not, polishing will help.
2- Filter your solutions (intra and extracellular)
3- make an external solution with high Calcium (5 to 15 mM CaCl2; sealing solution) and try to get your seals in that solution. then wash it out to lower Calcium soution.
Also, when trying to get the seal (applying negative pressure), if you see that your resistance is getting above 200 Mohms or so, apply a positive (+30 mV) continuous voltage and see if that helps.
check the osmolalties of your external and internal media. Internal having a 20 to 50 mOsm.kg-1 higher than external might help too.
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