Any experience in capturing Na+ currents in HEK293 or SHSY-5Y cell lines where the current is induced by ligand bonding and transport? Na+ acting as a symporter to the membrane protein which helps in binding the neurotransmitter and letting it into the cell. Na+ in, ligand in, a proton in and a K+ out. These generate small but detectable currents.
In my hands port-a-patch works one in fourty trials. I have checqued over 10 different cell types. Any advice from you will be welcome.
Thank you.
I use an EPC10 and follow the protocol suggested by Nanion. I will follow your suggestions.
Thanks again,
Ricardo
There is a book "Patch-Clamp Analysis advanced Techniques", chapter 14 "Planar Patch Clamping" , which I think would help you. It helped me a lot when I performed potch a patch system. I totally agree with David Baez-Nieto, that you should be careful when harvesting cells. If possible, try "tryple " from Gibco.
I have started using it for HEK293 cells and 2/3 times it was great capture and i realized that post transfections, instead of 48 hours when the confluency is 60-70%, and it may be 36 hrs ..its better to do the experiment. Yes I did face the same issue that when there is over confluency it doesnot capture well or the cell is lost. I do without trypsinisation and PBS washes without Ca2+/Mg+. Brief spin and suspension in the external solution. I am looking for Na+ currents. If anyone has a suggestion for capturing na+ currents, can let me know. right now we are using Nanion's solution only.
Take care that the dissociation procedure is very smooth. Tapping the cell culture dish harshly and vigorous pipetting of the cellsuspension will lead to pertuberances of the plasma membrane. This inturn decreases the success rate.
I am working with stem cell-derived cardiomyocytes and get nice success rates.
Hi Ralf, well I take care in cell dissociation. Till now I have just tried thrice and success is good. The input I am looking at is that does one want to take extra care while transfecting the cells and harvesting them at what point for the recording to avoid overgrown cells. I am talking about mammalian adherent cells now (HEK or neuronal types).
Hi Kalpita, did you try to contact the Nanion people? Usually, they have a very close relation to their customers and have a very good overview what was already done with their instruments.
Hi, Ralf.
My next step is that. One of the previous students in lab had used it for a cell attached mode and had some issues and could not pursue further and hence I started using recently and haven't contacted them yet. I will be checking with few of my own buffers as external solution. I am able to capture some current but we need to figure out how things can be performed better.
Thanks
I worked 6 years ago with the Port-a-patch device and we had no problems to get good Na+ current recordings with Nanion solutions from a stable expressing cell line. At the same time, we were using a "conventional" patch-clamp device to record the same current in the same batch of cells. Main problem for us was that the results obtained were significantly different (current amplitude and, mainly, activation/inactivation voltage dependence and kinetics), so we stopped using the Port-a-patch because we did not feel the results obtained with this device were reliable.
However, this was 6 years ago. I haven't use it later and perhaps many things about the Port-a-patch device are changed since then.
Hi there, does anyone have issues with getting the right resistance before even getting experiments started? We've had a few batched where the resistances were all in the giga ohm range but was apparently due to issues at production and now having the same issues with a new batch! Any help or advice would be greatly appreciated!!
- Badges
- Science method