difficulty in forming whole cell

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lazy
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difficulty in forming whole cell

I am recently experiencing a difficlty in forming a giga-seal and the whole cell configuration.

(1) I have difficulty in forming a giga-seal (many times only some hundreds MO).

(2) I can rupture the membrane, but the wlole cell has gone within one minute (mostly within some seconds.)

(3) I tried changing the program of lelectrode puller (Narishige) but no improvement. My electrode has 3-5 MO.

(4) Then, I prepare new pipette solution). The 1st patch was excellent (>40 min). Thus, I believed the pipette solution was the cause. But then after in the same day, the same problem came back.

(5) Cells (HEK) seem to be fine.

If you should have the similar experience, would you please give me advises? What should I do?

Thanks in advance.

lazy
lazy's picture
Thanks, Konstanz

Thanks, Konstanz

(1) Solutions
My bath solution (312 mOsm): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, % HEPES, 10 glucose

My pipette solution (293 mOSm): 130 KCl, 1 MgCl2, 10 EGTA, 10 HEPES.

Osmo was calculated, not measured.

Do you think they are correct or something wrong?

(2) Trypsin etc.
Passage with 0.25% Trypsin-EDTA at 80% confluence.
Also I use 200 ug/mL G418 (geneticin).

If you find something wrong, please give me your advises.
Thanks.

lazy
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Seems to be trypsin as a

Seems to be trypsin as a potential cause of difficulty. I will reduce it.
Thanks for your valuable suggestions. I appreciate.