I tried to record electrophysiological activities (eg: action potential) of neurons from cortical organoids at 100-day-olds using whole-cell patch clamp. I was able to get the giga seal easily. However, I cannot open the cell. Because the cells were quite small,attempted applying negative pressure to rupture the membrane would draw part of the cell into the pipette instead of rupturing it. So I tried to increase the resistance of the pipette from 6 MOhm (with positive pressure on) to ~8.5MOhm, and tried to rupture the membrane again. But still I cannot open the cell. I used potassium gluconate internal solution, and standard ACSF, recorded at 35 degree celsius, with flow rate ~2ml/min.
Hi Mai,
First thing, I will echo Max Anstötz here: try using the ZAP button to help with the break in. It is very effective most of the times. Have you ever used it?
Second, I would discourage you to increase the resistance. If the tip of the pipette is smaller, it'll be even harder to break the seal.
I had a preparation once that the difficulty of breaking the seal would increase with the amount of time I'd spend in cell-attached. Do you take too long to get the gigaseal? Try reducing this time to see if it improves.
Give us some feedback after you've tried some of our suggestion.
Good luck,
Andre
Hi Mai,
If the cells are small, firstly I recommend that you use smaller tip electrodes ~10 megaOhms. Secondly, once you form giga-seal, instead of suction, apply incrementing voltage or current steps (0.5 ms long at 1Hz) till you break the membrane, as all membranes will eventually break under such electrical challange (ie., electroporation). For details of the method, please see my previous publication for details about membrane electroporation, without using a dye. Once you determined the membrane breakdown potential threshold then you can use the same amount of voltage or current to succesfully break down membrane in a more controlled manner. I have developed this method in retinal whole-mounts a while ago, but I have been regularly using it in brain slices, cultured cells and human IPSC derived or organoid cells. Once you get used to it success rate is over 90%, and it is relatively simple.
best wishes,
Refik
Article Semi-loose seal Neurobiotin electroporation for combined str...
Hi! the pipette internal solution contained amphotericin B or nystattin. Perforated whole-cell current or potential recordings might be usefully tested.
Thanks a lot for all the answers!
We only applied small negative pressure to have the giga seal, and the giga seal was achieved easily and quickly. And we checked the osmolarity of both the internal solution (290mOsm) and the ASCF (304mOsm) which were in the normal range. So I don't think it's due to the osmolarity.
We tried to increase the resistance of the pipette because the cells were pretty small. With a resistance of 5-6 MOhm, the tip size was almost half of the cell body size. That's why we increase the resistance of the pipette to avoid sucking in almost the cell.
We also tried to use the ZAP button to open the cell but it didn't succeed.
I sometimes combine zapping with small continuous negative pressure until the membrane gives in. Always Start with the smallest zap (25 microsec) and increase to 50, 100...etc. works well with all sort of cells especially large cells such as smooth myocytes (from detrusor).
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating