I'm working with F11 cells (a DRG Hybridome cell line), and I'm trying to do current-clamp on this cells but I only can register RMP ~30mV, and this cells only spike a single action potencial. I don't know how I can solve this question. How can I work on the RMP value? And how can I increse the excitability of my cells?
I am not working with this cell line, but I guess something is wrong either with your culture conditions or with the internal / external solution.
I found this paper. Maybe you can compare their protocol to yours. Article Serum-deprived differentiated neuroblastoma F-11 cells expre...
Dear Ana Bernardo ,
Stefan Weigel has a very good point. Recheck your solutions (internal and external) to see if there isn't something unusual. Also, try an internal solution from a colleague to see if the problems persist - that's a good way to know where to start looking for issues.
Other tips:
- Pay attention to your leak right after you break in. If it is too high, the current-clamp quality will be affected and the membrane, depolarized.
Be very cautious with the gigaseal formation. Try to get a very good resistance in this step. If it is not >>1 GOhm and the leak in this step isn't close to zero (I like to aim for less than 20 pico Amps), the quality of the recordings will be questionable.
- Wash your cells well to remove all the serum before patching, which helps with the gigaseal formation. If you get a good gigaseal and very low leak currents, then the issue of the depolarized RMP should be your interal/external combination.
- Other details could influence the RMP, such as very low input resistance but I believe DRGs have relatively high input resistances.
- Liquid junction potentials could be an issue if they are not corrected, but only if the internal/external combination is aberrant and/or the input resistance is extremely low (
Consistently evoking single spikes is tied to unusually depolarized Vrest, which can be due to solutions, patch quality, or cells - as already pointed. Besides all the great suggestions, I can only add the following.
- After the giga-seal establishment, waiting some time before the break-in is good to stabilize the cell and the seal.
- You can measure Vrest on-cell to rule out internal solution or whole-cell quallity - granted good seals. The two approaches I know of include:
* measuring directly from on-cell current-clamp (however, this can give depolarized Vrest by ca. 20 mV): Article Cell-attached current-clamp recordings from cortical neurons
;* estimating from on-cell single-channel, i.e. using I-V profiles of NMDARs: Article Membrane Potential of CA3 Hippocampal Pyramidal Cells During...
.Alternatively, APs could be elicited on-cell, if leaky seals are already ruled out. If you consistently evoke single spikes even with strong stimuli on-cell, it's either the external solution or cells.
Best,
Bogdana
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