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difficulty in forming whole cell [View Printable]
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lazy
Group: Member
Posts: 17
Joined: Apr 03, 2008
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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.
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| Posted Aug 25, 2008, 8:50 AM |
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Konstanz
Group: Member
Posts: 15
Joined: Aug 07, 2008
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At least with HEK cells, the problem could be the osmomolarity of your solutions. Extracellular solution should be around 290-300mOsm, intracellular 285-290mOsm. Say me if it helps :) |
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| Posted Aug 28, 2008, 8:09 AM |
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Konstanz
Group: Member
Posts: 15
Joined: Aug 07, 2008
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By the way, another problem could be the over-tripsinisation. |
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| Posted Aug 28, 2008, 8:58 AM |
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lazy
Group: Member
Posts: 17
Joined: Apr 03, 2008
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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. |
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| Posted Aug 28, 2008, 12:10 PM |
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Konstanz
Group: Member
Posts: 15
Joined: Aug 07, 2008
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(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.
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Osmo more or less OK. But it's better to measure it, in order to exclude some surprises :) As for the trypsination - the question is the time - how long (we use 0.01% Trypsin, which works, for 2-4 minutes). Another important question is the Ca2+ inside the pipette solution. Do you have any? By the way, not for sealing, what's about ATP? |
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| Posted Aug 29, 2008, 2:58 AM |
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lazy
Group: Member
Posts: 17
Joined: Apr 03, 2008
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Seems to be trypsin as a potential cause of difficulty. I will reduce it.
Thanks for your valuable suggestions. I appreciate. |
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| Posted Aug 29, 2008, 10:51 AM |
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