I'm doing some perforated patch-clamp on human-derived IPS cardiomyocytes. Most of the cells die (by exploding/melting in vesicles) as soon as I touch them with the tip of the glass pipette. Does anyone know why this happens and how to solve this issue?
[two ideas I had: reducing the concentration of Amphotericin B in the internal solution, changing the composition of the glass pipette]
Hi Dario,
The first thing I would do is check the osmolarity of my medium. If the cells rupture when you touch them, it may be because your medium is hypoosmotic and the cells take in water to rupture point. Also check that you fill the tip of your pipette with medium enough to prevent amphotericin B to reach the tip before you achieve a good seal. When you back fill be careful to not put too much pressure which would result in your pipette leaking amphotericin B. This said I do not think AmphoB is the culprit, it happened to me to have amphoB leaking by the tip, what it does is it makes impossible to get a seal and clamp your cell which ultimately dies. It never made the cell explode. I would also check the osmolarity of your pipette solution. cell exploding is generally due to hypoosmolarity in the surrounding medium and the IPS cells have a fragile membrane compared to primary cardiomyocytes. Also to improve the patching you may want to incubate your cells with 10uM BAPTA-AM for 10 min (that's what I used on primary cardiomyocytes, worked very well) before washing it off and patching. That will prevent your cell from contracting when you patch which result in shredding the membrane.
Hope that will help.
I would say try different concentration of Amp B (dissolved in DMSO) in internal solution. High concentration might do that. To conform the cell died from Amp B, you could also use beta-escin which is water soluble. Also the speed and angle you approach to cells matter. Check these things. If you either able to solve problem or not, do write here again.
Good Luck
Chandra
Does this happen only with AmpB or does it happen regardless? How much AmpB do you use? Anyways, I would first try without AmpB and see how it goes. I mean, that the cells do not die on contact, just to eliminate AmpB from the equation. Then if you still have the same problem, think about the osmolality of you IM and backfilling...etc (as advised above). You could also try different setting for your patch pipettes (slightly smaller pipettes tips maybe, although it may increase the time to achieve PWC mode). I find it helpful to have a 20 to 30 mOsm.kg-1 difference between the IM and EM (IM being hyposmotic). Cells must look nice and healthy (nicely transparent cells, no obvious wrinkles on the membrane...etc). All the best.
I forgot, the first thing to check is if you have vibrations, the tiniest of vibration can shred the membrane when the tip touch it and the cell would die. Also verify that you do not have drift in your micromanipulator. Hope that may help
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