h*******g
发帖数: 711 | 1 Currently we are studying the function of a channel by exogenously express
it into HEK293T cells. However, in our HEK293T cell-line, it seems that
the background current is so huge, ~10 nA. I'm wondering whether your guys
also observe this phenomena? | d********r
发帖数: 3279 | 2 no way. there is almost no background currents in HEK cells. You must did
something wrong on your recording setting.
【在 h*******g 的大作中提到】
: Currently we are studying the function of a channel by exogenously express
: it into HEK293T cells. However, in our HEK293T cell-line, it seems that
: the background current is so huge, ~10 nA. I'm wondering whether your guys
: also observe this phenomena?
| i***R
发帖数: 663 | | h*******g
发帖数: 711 | 4
HEK cells. You must did
I know there couldn't be. Nobody shows a big current
in any paper. However, it is the case at least in our
cell-line.......
【在 d********r 的大作中提到】
: no way. there is almost no background currents in HEK cells. You must did
: something wrong on your recording setting.
| h*******g
发帖数: 711 | 5
transfected NMDA subunits with Glu/
current?
No, actually, we did not do any treatment on the
cell, and just record IV from -100 to 100. | h*******g
发帖数: 711 | 6
Not really, cos there is a reversal potential less
than -40 mV observed in the IV curve. I don't think
leak can do this. Besides, this current is vanished
after transfected with our target channel......
【在 i***R 的大作中提到】
: Bad seal?
| i***R
发帖数: 663 | 7 Do you think K is charge carrier or Cl ?
Why don't change your ex solution composition to figure out.
Still, 10nA is Very high. We never see such current in blank HEK cell.
There is 100pA background current at most.
If you are not sure about your HEK cell. The easiest way is to get some
other HEK cell from other lab or ATCC.
【在 h*******g 的大作中提到】
:
: Not really, cos there is a reversal potential less
: than -40 mV observed in the IV curve. I don't think
: leak can do this. Besides, this current is vanished
: after transfected with our target channel......
| h*******g
发帖数: 711 | 8
】
figure out.
current in blank HEK cell.
easiest way is to get some
First, I would like to thank you for your good
advice. I have used NMDG+ MSA solution. However, the
current is still there, which is really wired.
Anyway, we have ordered a new line from ATCC. Hope
this can solve the problem.
【在 i***R 的大作中提到】
: Do you think K is charge carrier or Cl ?
: Why don't change your ex solution composition to figure out.
: Still, 10nA is Very high. We never see such current in blank HEK cell.
: There is 100pA background current at most.
: If you are not sure about your HEK cell. The easiest way is to get some
: other HEK cell from other lab or ATCC.
| l******8
发帖数: 1691 | 9 That's very confusing.HEK cell should not behave like this.Most likely there
is something wrong in the setting, not in the cell line.
First of all, you said the Vr is < -40 mV, assuming the Vr is -50 mV, so, at
-50 mV, you have 0 current, right? Then, at what holding voltage did you
get a -10 nA current? You said this is the background current, so I assume
it is not depolarized.
What is your noise level (peak to peak)? Also, it would be helpful to know
your depolarizing step size during membrane test, and the resulting current
size.
【在 h*******g 的大作中提到】
:
: 】
: figure out.
: current in blank HEK cell.
: easiest way is to get some
: First, I would like to thank you for your good
: advice. I have used NMDG+ MSA solution. However, the
: current is still there, which is really wired.
: Anyway, we have ordered a new line from ATCC. Hope
: this can solve the problem.
| h*******g
发帖数: 711 | 10
like this.Most likely there
cell line.
assuming the Vr is -50 mV, so, at
holding voltage did you
background current, so I assume
would be helpful to know
and the resulting current
Thanks for your comments.
First of all, we are not handling the voltage-gated
channel. Plus, it seems that the background current
is not voltage-dependent because the IV-curve is
linear. The 10 nA I mentioned is under a holding
potential of 80 mV.
The noise level in our system is about 2 pA,
measured by the Irms. And in the membrane test, I
use a pulse of 2 mV and the series resistance
measured is about ~10 MΩ, so we can calculate that
the current is ~500 pA.
【在 l******8 的大作中提到】
: That's very confusing.HEK cell should not behave like this.Most likely there
: is something wrong in the setting, not in the cell line.
: First of all, you said the Vr is < -40 mV, assuming the Vr is -50 mV, so, at
: -50 mV, you have 0 current, right? Then, at what holding voltage did you
: get a -10 nA current? You said this is the background current, so I assume
: it is not depolarized.
: What is your noise level (peak to peak)? Also, it would be helpful to know
: your depolarizing step size during membrane test, and the resulting current
: size.
| l******8
发帖数: 1691 | 11 I have many questions.It seems that you do not have an experienced
electrophysiologist in your lab to consult
Maybe you have a good reason, but why do you hold the cell at 80mV, instead
of something like -60mV? You are whole cell clamping, not in cell-attached
mode, right?
When the other person asked you whether it could be a bad seal, I think you
should say what your seal resistance is, instead of inferring from your Vr.
【在 h*******g 的大作中提到】
:
: like this.Most likely there
: cell line.
: assuming the Vr is -50 mV, so, at
: holding voltage did you
: background current, so I assume
: would be helpful to know
: and the resulting current
: Thanks for your comments.
: First of all, we are not handling the voltage-gated
|
|
