Going whole cell problems

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egovoru
egovoru's picture
Going whole cell problems

 I would like to measure whole-cell currents in HEK cells. I have no problem achieving a GOhm seal, and sometimes I also succeed in going whole cell. But, mostly, when I try to rupture the membrane, the seal breaks. I used both suction and the ZAP function of my Axopatch 200B amplifier, to no avail. My pipette resistances are btw. 4 and 5 MOhms, as these are supposed to be optimal for whole-cell recording. I would be very grateful for any suggestions how to solve this problem.

The FFM
The FFM's picture
what are  the molecular

what are  the molecular compositions of your extracellular and pipette solutions and what are the measured osmolarities of each solution when they are prepared?

If you have too large a difference in osmolarity between the two solutions you will have difficulty keeping a seal.  However you will need a slight difference between the osmolarities of the two solutions

Most physiological solutions are between 296-330 mOsm

You can check the osmolarity of your solutions using a vapor pressure osmometer - these devices are made by companies such a Wescor.  Pipette solutions should normally have an osmolarity about 5% less than the extracellular solution.

E.g pipette soln 295 mOsm

 Extracellular solution 310 mOsm

you can adjust the osmolarity of your solutions by adding an appropriate amount of sucrose.  If the difference between the osmolarities of your solutions is significantly more than 5% you may well have problems keeping a good seal.

egovoru
egovoru's picture
 Thank you very much for your

 Thank you very much for your response. My solutions are as following (in mM):

Pipette:

KCl 126
MgSO4 2
CaCl2 0.5
Na-EGTA 5
HEPES 25
pH 7.2

Bath:

NaCl 150
CaCl2 1.8
KCl 4
MgCl2 1
glucose  5
HEPES 10
pH 7.4

Unfortunately, we don't have an osmometer in the lab. The calculated osmolarities of my solutions are ~297 and 331 MOsm, respectively, although I understand that these cannot be really relied upon. Today I tried adding 20 mM glucose to the pipette solution (i.e., made it ~4% difference, if to believe the calculated values), and it indeed seemed to improve the seal stability after the membrane rupture. However, it decreased the frequency of formation of the seal itself.