troubles with patch clamping at 37 degrees C

5 posts / 0 new
Last post
green_bear
green_bear's picture
troubles with patch clamping at 37 degrees C

 Hi all,

I'm having difficulties patching at 37 degC. My pipette appears to drift back and forth, so it's really hard for me to get a stable giga-seal. If I turn off the heat and wait for the solution to get back to room temperature, the problem vanishes. 
Any idea on how to prevent or minimize the drift? I'm using a stage heater. Would inline heater be better?

I'd appreciate any help on this.

Cheers,
g_b

lazy
lazy's picture
I have tried some in order to

I have tried some in order to get rid of  this problem. Note that all manipulators give a drift with a high sensitivity to higher temperature. I do not recomend to try to achieve G seal at 37C because of the reson shown previously.

(1) G seal at room temerature, whole cell, and them go to 37C.

(2) G seal, lift the cell from coverslip, go to 37C, and whole cell.

I prefer the option (2), since option (1) will get suffered with severe drift again. While, the option (2) is free from the drift problem, and even the whole cell would be kept longer, the cell swelling/shrinking easily detactable. 

You may be required to get used to the technique to lift the cell.
Hope this usefful.
 

green_bear
green_bear's picture
 Thanks, lazy, for your

 Thanks, lazy, for your prompt response.
By "lift the cell from coverslip", do you mean completely detach it from the coverslip? Would that be a problem if my cells attach strongly to the coverslip? Is there a time window after seeding the cells when I should start patching them so they won't attach too much? (say, for HEK cells)

Thanks,
Hung.

lazy
lazy's picture
(1) Yes. I completely detach

(1) Yes. I completely detach the cell from the coverslip. Of course, the cell should stay inside the bath solution even lifted .  So, you are free from the drift.

(2) Do you use poly-lysine coated covereslip?

  1. I have used cell suspension solution instead of  seeded cells on the coverslip.
  2. You stop the perfusion system.
  3. Then you put a few drops of cell suspension into the recordeing chamber.
  4. Wait until the cell reaching down to the bottom.
  5. You can see it with microscope. The cell must be round. You may feel the cell very small.
  6. Wait for a while (a couple of minutes - some minutes) until the cell slightly attached to the bottom glass, depending on your recording system.
  7. Access the electrode for the G seal.
  8. Once the G seal was achieved, then slowly lift the cell.
  9. Go to the whole cell configuration.
  10. Start your perfusion system with temperature of 37C.

I have succeeded this with both CHO and HEK cells. There must be different ways of cell lifting method which you may find elsewhere.

Hope this usuful.

green_bear
green_bear's picture
 Thanks again. I used to try

 Thanks again. I used to try patching cells early after they are seeded (when they still remain rounded). However, it is usually much harder to patch when they are not attached because they might move as the pipette presses on them... Right now I'm using aclar coverslip, but I used poly-lysine-coated glass coverslip before (cells indeed tend to attach better with it).
I guess I should give your suggestions a try, although it might be problematic for other cell types that I'm also working with are way bigger than HEK cells (such as fibroblasts). Also, sometimes we need to stamp the cells together in certain pattern, so lifting them off isn't an option...

g_b