Hi Po-Han,

Whole-cell patch clamping of virus infected neurons is similar to recording from any other cell or neuronal culture. First of all you should make sure that your external and internal solutions are good to record from normal and healthy neurons. Cultured cells are often vulnerable and require gentle care to throughout all the procedures (such as changes of media) and also during the transport and transfer of cells to recording chamber. Another important point is the potential infections by other bacteria/pathogens or pore forming toxins/antibiotics that can deteriorate cellular health and viability.

Best wishes,

Refik

You may need to check internal and bath solution, particularly potassium concentrations. If bath solution contains high K+ concentration, the resting membrane potential may become depolarized. Or, if internal solution is low, the resting membrane potential would be changed as well.

Hi Po-Han,

you can try to lower your virus concentrations, you can make some test using different concentrations.

Why your internal solution is ATP and GTP free?

Have you checked the osmolarity of your solutions? You have the same problem even in non infected cells?

Hi all,

Thanks for your answers.

For maintain the cultured neurons, Do I need to do half change all the time. For example, after glial inhibition or virus infection, do I also need to do half change the medium? Will it cause some FUDR or virus left inside the medium and damage the cells?

Can I use antibiotics to prevent any contamination? I never use any antibiotics in the cultured neuron. I heard it will kill the neuron.

Because our culture room and patch-clamp setup is very far. I always transfer the coverslip with neuron culture in to a plate with tyrode solution. and transfer from plate to recording chamber again under microscope. Do you guys have any suggestion for the solution I should use for transferring between culture room and patching setup.

Our osmometer is broken so I cannot measure it. But my solution works very well for new cultured neuron within 1 weeks. This is the reason I think the solutions have no problem. And probably because they are much healthier. However, it is very hard to get good patch on 2 weeks old culture. I need time for virus expression. But my culture is not very healthy in second week.

For extracellular Tyrode's solution, it is HEPES base solution with 2mM K+, 125mM Na+, 135mM Cl-. I adjust the pH to 7.4 with NaOH.

For internal solution, I didn't add any ATP or GTP is because I am not planing to do any long term recording. I am not sure whether that is the reason because membrane potential is high right after break in. Do you guys think ATP is critical for short term recording in the culture system?

Thanks again for your response. I am very appreciate.

Hi Pho,

I think you should add antibiotics to your media. In my experience, antibiotics did not kill the neurons. But I used 5% antibiotics(Pen/Strep) instead of 10%. For transferring cells from the cell culture room to the patching set up, I used fresh neuron culture media. Another trick that you can try is to re-plate the cells the day before you patch them.

It is also important that cells need to be "healthy". Otherwise, it is hard to make a good seal (i.e., greater than 1 gigaohm). In some conditions, surface membrane on "transfected" cells would become "hard" and rigid. Consequently, it almost can not make any good seal at all.

Hi Po-Han,

I know it's an old post, but did you ever get an answer to your question? I'm encountering problems similar to what you described, and I'm wondering what the solution is. Thanks.