I have recently started to work with B50 neuronal cell line. The cells grow and proliferate well, but they do not exhibit any signals when I try to record action potentials from them using patch clamp (after up to 10DIV). The cells dont exhibit any sodium currents as well. I am doing the recording in DMEM with perfusion, at 35 degC.
To mention a few details, somehow the rest membrane potential of the cells range from -20mV to -35mV only and the cells look pretty different than those from primary culture. I am starting to wonder if such immortal cell lines express any sodium channels that result in action potentials. Does anyone have any experience with electrophysiology on cell lines?
Thanks!
Thank you very much, Ulrich. Thats very useful.
I grew the cells till passage 8 with 10% FBS (according to the cell line catalogue), and after that I am growing them with 1% FBS (in DMEM). Do you think this makes any difference, that is should the cells be grown in serum free medium right from passage 1?
Also, what extracellular medium were you using for patch clamp recordings? Was it nerobasal / DMEM or something else? We tried patch clamp recording with a perfusion of ACSF, but cells dont survive very long. Another thing, does the intracellular medium for cell lines have to be different from that used for brain slices/primary cells?
Another small thing for recording from cell lines, how important is the temperature of the recording chamber/perfusion medium? Does it have to be at 37degC or room temperature?
If you have any of your publications in regard to electrophysiology on neuronal cell lines, could you please send me a link to that? Thanks!
Hi Vini, generally speaking, if you are searching a cell model for neuronal action potential, none of the immortal cell line is that great as a model. You can push them to differentiate but they rarely resemble true neurons in their membrane properties. There is a reason why people are still using primary neuronal culture, and even in that case, you will realize even the primary neuronal culture is only an adequate model of some immature neurons and behave very differently from neurons you get from a brain slice from an older animal. If it is only the sodium channel that you are interested in, heterologous expression system is still better.
I have patch undifferentiated NG108-15 cells and if my memory serves me right they don't fire AP. But they will fire if treated with barium. Not very physiological I know.
Hi, Vini, I wonder what the junction potentials may look like when you found the Vm ranged between -20 to -35mV for your cells, which recorded in DMEM as the perfusion.
Thanks for this doubt, Desheng! I must check for the junction potentials! I must also mention that when I started recording, I was using ACSF (which my colleague uses for brain slice experiments) and the Vm used to be ~ 40mV (better than DMEM though). I had to switch to DMEM because the cells somehow looked very unhealthy in ACSF perfusion..
Hi, Vini, Honestly, I have no any doubt about your recordings but I am just thinking there may be some differences in junction potentials between ACSF perfusion and medium (DMEM) perfusion. On the other hand, pH for ACSF perfusion needs to be air-buffered while DMEM perfusion, which generally is HEPES-buffered, may NOT need that. Thirdly, there may be some differences in the temperature between the incubator (37celsius degree) and patch clamp recording (room temperature or 32C or 35C). You know, all these factors may affect the recordings. That is the only reason I wonder if you could kindly share the information for junction potentials. Feel so sorry for the misunderstanding and confusion that my comments led to...
Hi Vini,
Just want to double-check: have you done your recordings in current clamp or voltage clamp mode? In current clamp, you have to use the DC stimulating current to set the Vm negative to -60 mV. I would expect that at -35 to -20 mV, all the Nav and Cav channels will be inactivated and no spike can be elicited if even the inward-current generating channels are expressed in the membrane. Similarly, when you try it in patch-clamp mode, you have to set Vh negative to -80 or -90 mV to remove SSIN and be able to check-up for availability of inward Nav or Cav currents.
The external saline solution should contain about 140 mM Na, 5 mM K, 1.8 mM Ca, 1 mM Mg and some glucose (~10 mM). There are a few commercially available buffers which meet these requirements. For Cav currents it may be important to have ATP/GTP in the internal (patch pipette) solution and free [Ca]in set at
An obvious correction/comment on what I said above: to separate currents in voltage-clamp mode, Cs salts should be used in internal solution instead of K salts. In current clamp mode, KCl is a primer choice for pipette solution for AP recordings