Hello everyone,
I am trying to record single channel events on my HEK293 cells stably expressing Cav2.2 channels, using the cell-attached patch clamp technique.
I used the following solutions:
Extracellular: 150 KCl, 10 HEPES, pH 7.3 with KOH
Pipette solution: 110 BaCl2, 10 HEPES, pH 7.4 with KOH
Unfortunatelly, I haven't been able to find the channels so far.
- I want to step the cell to 0, 10, 20 and +30 mV from a holding potential of -100 mV. I understand that to hold the membrane at -100 mV I have to set the V-command in my protocol to +100 mV (and steps to 0, -10, -20, and -30 mV). Is this correct?
- In that case, the currents I expect are patch inward currents (barium ions flowing from the pipette into the cell). I understand these currents should appear as outward currents (positive) in my recordings, is that right?
- Should I invert the polarity of both voltage and currents for analysis and representation?
- Finally, I am trying to use the Patch mode in my Axopatch 200B amplifier, but every time I try this a regular spike noise will appers. Do you know why this might be happening?
Thank you very much for your attention!
Diego
Hello Diego Fernández-Fernández
I am also trying to get some single channel recordings of the Nav.
The solutions look fine in my opinion, but the protocol in my opinion is not correct.
The holding that you have in figure 2 is 0mV and not +100mV. If I am not mistaken in the Clampex software when you create a new protocol you decide the holding potential under the option outputs and there you put the desired holding.
Afterwards in the option waveform you can decide the first step and the delta level.
And yes the current you expect to see in the raw recording should be "positive".
About the regular spikes in the Patch mode, I was also wondering and decided to ask the Axon support and this was their reply "Instead of resistor, a feedback capacitor is used in the Patch mode in Axopatch 200B amplifier. The voltage across the feedback capacitor cannot ramp in one direction forever. At some point the capacitor voltage will approach the supply limits and the integrator must be reset to start again near zero volts. Thus the current record must be interrupted for 50 us while the integrator and differentiator reset. The frequency of resets depends on the current passing through the headstage, with the larger current requiring more frequency resets."
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioecology
- Systems Ecology
- Landscape Ecology
- Connectivity