Whole cell patch clamping TE671 cells

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Hakonan
Hakonan's picture
Whole cell patch clamping TE671 cells

 Hi folks!

I am trying to get whole cell configurations on TE671-cells (measuring Acetycholine receptors). As I have some problems on getting whole cells, you may help me or give me some advice.

1) Once in the bath solution my TE671 cells get vacuoles after some time (sometimes even after 15min). First I thought this might be due to the osmolarity of my bath solution, but after adjusting glucose concentration, it was still the same (I don't have an osmometer). I am using DMEM as culture medium with trypsin (for about 1min) to get them detached.

My bath solution:
150 NaCl
2,8 KCl
10 HEPES
2 MgCl2
2,5 CaCl2
15 Glucose

My pipette solution: 4 NaCl
144 KCl
10 HEPES
2 MgCl2
2 ATP
10 EGTA

I also tried not to centrifugate them, on the assumption that this might damage them. But this also wasn't a success. I wonder if that is just the way how the cells are? Have any of you had the same experience with TE671 cells?

2) I have problems with the break-in after my gigaseal. The sealing is not the problem (sealing rate between 60 and 90% of my cells with seals between 10 and 20 GOhm), but when I try to get the whole cell configuration, I just achieve membrane resistances about 200MOhm with Access resistances under 10MOhm. Just in about 5% I get whole cells with membrane resistances above 1GOhm. My Whole cell rate is too less I think. I also used pipettes with lower resistances (about 2MOhm), even fire polishing them. I used mouth suction and suction with a syringe, having more success with the second. Any suggestions how I can enhance my whole cell rate?

Thank you very much for your help!!