I want to use 10mM BAPTA in k-gluconate-based internal solution for whole-cell patch clamp recordings but I have difficulties to dissolve it.
I know it is possible to add BAPTA (acid) directly to the internal solution by adjusting the pH (with KOH) or dissolving it in 0.3N sodium bicarbonate before adding it to the internal solution.
I would be very thankful if you could share your experience!
Hi Silvana,
I would simply add the BAPTA acid to the solution, even if it does not dissolve at first, and subsequently adjust the pH with KOH. At some point in this process the BAPTA should dissolve just fine.
If this does not work, you might want to give some more information on what you are doing, e.g. do you use K-gluconate or do you use gluconic acid and add KOH, also which pH buffer do you use (HEPES?).
Hi Christian,
Thank you for your answer!
I use K-gluconate (122mM) and HEPES (10mM).
I succeeded to dissolve the BAPTA by adding KOH.
Now the problem is that with this BAPTA-internal solution the gigaseal seems impossible.
I was wondering is there any difference between 1) dissolving the BAPTA in intracellular solution ready for patch (pH already adjusted with KOH), then adding the BAPTA and adjusting the pH again; and 2) adding the BAPTA before any pH adjustment is made and add KOH at the end.
Hi Silvana,
I would dissolve it directly in your pipette solution and you could use some brief sonication to help it dissolve completely. Since you are using a K-gluconate solution I would also recommend to use BAPTA tetrapotassium salt next time.
Hope this helps,
Ezequiel
Hi Silvana,
I don't think that it makes a difference at all, since obviously the solution you end up with will be the same, as long as the same pH is established.
If you use potassium salt of BAPTA and K-gluconate, you will probably have a basic solution and will have to add acid to adjust the pH (but this depends somewhat on the buffer, whether you are using the salt or the acid (or base) of the buffer, and on its concentration). So you will end up with a slightly different K+ and (when using gluconic acid for the adjustment) gluconate concentration.
It is a bit difficult to answer your question without more information about your intracellular solution, e.g. is it completely Ca2+ free or are you aiming for a free concentration in the physiological concentration, what else is present (Mg2+ , buffer HCO3-, HEPES or something else? At which concentration?), your bath solution may play a role.
What you might try is some kind of tip filling technique, the simplest being applying some negative pressure / suction to the pipette after entering the bath to fill the tip of the pipette with a little bit of bath solution. After establishing the whole-cell configuration (I think this it what you are planning to do?) you will have to wait a little bit longer than usual until the pipette solution has equilibrated with the cytosol of the cell.
Hello Silvana,
I too use a K-glu internal solution (130mM) and have found that to dissolve 10mM BAPTA into solution you need to slowly heat the solution up. I then add the remaining solutes (NaCl, HEPES, NaATP/NaGTP) pH and configure the osmolarity for the solution.
With this method i have had no problems forming gigaseals with brainstem cells.
Hello Silvana,
I have little experience with BAPTA, but a lot patch-clamping cardiac myocytes. I also found very difficult to seal with BAPTA in the internal solution, and after some discussions with different colleagues we conclude that it is challenging to have good seals with no calcium in the pipette solution. It may be necessary for the correct contact between the membrane and the glass.
The only solution for this problem is to have very good cells and a lot of patience.
Good luck,
Oscar
it was not a problem for me even at 20 mM. you may try sonication if you have one around.
Thank you all for the useful tips!
Indeed, dissolving the BAPTA directly in the intracellular solution is easier with sonication and it requires some extra KOH. I might try BAPTA tetrapotassium salt next time (as suggested by Ezequiel).
Hi Ming, I didn't really fix the problem but just tried to target very good cells so the seal was better but I still had some problems.
Hello Silvana,
I use the same technique for dissolving BAPTA as you have, and it works fine for me. Just to make sure, have you checked the osmolarity? If you've added 10mM BAPTA (+ KOH) to a normal internal solution then it will probably be hyperosmotic.
I found BAPTA tends to make sealing harder, too. I found gigaseals fairly easy with 10mM on cerebellar Purkinje cells, but they are quite easy to patch. With cells that are harder to patch or higher BAPTA, it can be more of a problem. The obvious and simple aids are anything that makes patching easier generally, such smaller diameter pipette tips, searching for very healthy-looking cells, cells nearer the slice surface, and so on.
I have the same problem with this tread. I think your suggestion is the easiest way to do. I would like to try, may I know for how long I need to sonicate the solution Victor Z Han? I used the same concentration as yours which is 20 mM. Thank you very much in advance.
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