Dear patch-clamp enthusiasts,
For patch-clamp recordings of vomeronasal sensory neurons, I need a cesium-based intracellular solution. Because I also need a defined amount of chloride, I have to substitute with gluconate. For the last few days, I have been trying to use gluconic acid for this, but the pH will not stabilize properly, only after 20+ hours... And when it finally does, my osmolarity is off the charts because I had to add (too much) CsOH to reach the pH...(My goal is pH 7.1 and 290 mOsm)...
Is there some sort of trick with gluconic acid or thing I can do to improve the pH stabilization? I already picked the brains of our technicians and checked old lab books, but everything checks out. I really appreciate any help you can provide. :)
gluconate precipitates calcium, very dangerous for cell recordings, you never change only one parameter, do you know that?
Thanks you very much for your answer! What would be your suggestion? I’ve already used K gluconate based solutions and had no problems whatsoever. And I've already had a solution with less gluconic acid (93.5 mM) and more chloride and it was fussy but manageable...It seems that this amount of gluconic acid ( 115 mM) is a problem...
I am using solution which contains Na gluconate, K gluconate and Cs Gluconate; of them first two are added as ready-made salts, only 40 mM of CsOH are titrated with gluconic acid. Under these conditions everything works fine with the end concentration of gluconate 140 mM (I have very low Cl concentration). For titration, I am using pre-made 1 M stock solution of gluconic acid, because as a dry substance gluconic acid exists in form of gluconolactone and requires considerable time to open cycle and become really acid. In a solution, at least part of it is in acidic form.
Now what is also important, acidic evironment inhibits this convertion from lactone to acid, or, rather, shifts equilibrium point to lactone. So, if you add first the acid and titrate with CsOH, you have initially mostly lactone, neutralize it with your added CsOH, pH shifts to desired value, but after it some more lactone becomes acid, pH becomes acidic and you have titrate again, and this cycle goes very long.
It is therefore better to add first CsOH in desired concentration and then titrate (slowly) with pre-made solution of gluconic acid. Under these conditions it is added to basic solution and is converted relatively rapidly.
One more possibility is to make once Cs Gluconate stock solution beforehand by such titration and save time for a day when you make a pipette solution, but this I never tried.
Viatcheslav V. Nesterov Dear Dr. Nesterov, thank you very much for your answer and suggestions! I'm using a 2.55 M gluconic acid stock solution and started a new batch of my intracellular solution yesterday evening, where I was adding it, as you also suggested, at the end and titrated it slowly until I reached my goal pH instead of putting it all in at the beginning. For the future, I might switch to a 1 M stock of gluconic acid so that the pH changes are more subtle. Thanks again, and have a nice weekend! :)
Hi, Victoria! Using the acid may be a hassle... We use the cesium gluconate salt directly in our internals. Hello Bio sells it, but we make our own in the lab. Although simple, it takes some time (days) to make it. Let me know if you're interested and I can share the protocol. Good luck!
Andre Dagostin Thank you very much for your suggestions! I would really appreciate it if you could share the protocol. :)
Andre Dagostin, can you share it with me too, please? Thanks in advance!
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