You will need specific solution because the concentration of the different ions (Ca2+, Na+, Cl-, K-...) is crucial for neuronal activity recording. Check literature, but it should be something like this: (mM) NaCl, 140; KCl, 5; CaCl2, 2.5; MgCl2, 1; HEPES, 10; glucose, 10 (pH 7.4).

Hi Arwa,

The composition of your external solution depends on what you are trying to measure. In principle, you can do patch clamp in neurobasal medium if you like - keeping in mind that the presence of serum is usually not helping at all to get the giga-seal... However, as Daniel mentioned, if you want for example to specifically record calcium currents, or sodium currents, etc... you will have to use a well defined and appropriate medium.

If you tell us exactly what you would like to do we can certainly help you with the experimental conditions to use.

Best, Norbert   

Dear Arwa,

I have not done much patch clamping--only enough to try out the technique for single cell PCR.  However, the Faculty Member that mentored the project taught me that making sure that the osmolarity of your solutions is also critical.  She had an osmometer, and the lab would always check the solutions before use.

So, I suggest that you also check what the osmolarity should be for your particular neurons.

Good Luck!

Jill

Certainly you need to choose composition of your bath solution according to what you intend to measure. Culture medium is always a bit "sticky" and causes troubles with getting gigaseal, as Norbert mentioned. When we patch neurons which were for prolonged time in medium, and we have problems to get good gigasela, sometimes we even wahs them with a trypsin solution to clean the membrane. This must be doone very carefully, for some second only and with a trypsin solution diluted to 0.005%.

I used to patch in culture solution in DMEM and also in neurobasal medium. Neurobasal usually for serum-free culture, For DMEM I did not use serum in the patch solution, even if the cells were cultured in 5% serum. I also added 10 mM HEPES to the culture solutions to maintain the pH around 7.4. (In the incubator the CO2 is doing that, outside of the incubator something has to set the pH) It is true that the choice of the external (and also the internal) solution depends on the application. For me the cells survived longer in culture solution, but for specific ion channel currents and pharmacology I had to use defined (ACSF) extracellular solution with known ionic composition. The osmolarity is VERY important! The osmolarity of DMEM is around 310, the same as the ACSF in the books. For that the 276 - 280 internal solution (as written in books) is good. BUT the osmolarity of Neurobasal is around 280, thus you have to decrease (dilute) the osmolarity of the internal solution by about 10%. There are culture media for adult cells with osmolarity of 230! Seal formation in cultures usually easy. I do the approach visually, positive pressure outside the medium, go to cell, compensate junction potential, touch the cell (you see the deformation of the membrane, the positive pressure is pushing the membrane away) The seal is there when you release the pressure, even with minimal suction. I move a little bit back with the electrode not to deform the cell. If problems with the gigaseal, Check internal solution (Ca free, filtered), also electrode resistance. With higher resistance electrodes seal formation usually easier, breaking the cell membrane more difficult.