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...why EGTA in the pipette?

wanderer36


Posted 1/3/2008 10:59:44 AM
Halo!
...I had saw that most of the times people used EGTA in the solution for the recording pipette in Patch Clamp, so I would like to know for what is that EGTA and if there is so important.

Danke!

frasermoss


Posted 1/3/2008 6:14:57 PM
EGTA = Ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid

It is a chelating agent useful for the determination/buffering of calcium in the presence of magnesium.

It is NOT to be confused with EDTA - Ethylenediaminetetraacetic
which is a calcium chelator which will also chelate Magnesium and other divalent cations.

EGTA is included in the patch pipette for whole cell patch clamp recording to buffer fluctuations in intracellular [Ca2+] during recording. This prevents contamination of the recorded ion channel signal by Ca2+ dependent second messenger signaling pathways (e.g. calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of certain channels) which could be stimulated by the activation of the ion channel under study. Furthermore, you rupture the cell's membrane during whole cell clamp. It will become very unhappy and release Ca2+ from the intracellular stores in response to the insult. The EGTA will buffer large fluctuations in the intracellular [Ca2+] and make it easier to maintain a good seal during the recording. There are protocols that do not include EGTA in the pipette, but under these conditions it is very hard to maintain the seal.
wanderer36


Posted 1/4/2008 10:50:46 AM

...it sounds a good explanation, thanks.

Anyway, if we want to see the Ca++ component of some currents, we have to avoid to used EGTA, or not? For example, when we study the currents throught nicotinic receptors, wich are currents for both, Na+ and ca++, and we want to see how much is implicate each ion in the wholle current, then could EGTA introduce some alterations? Or when we want to see the activation of channels that need Ca++ to become open, like BK channles...

Thanks...

frasermoss


Posted 1/4/2008 6:30:31 PM
As an example for BK patching you can have 10mM EGTA in your patch pipette solution and on the day of recording add a set concentration of fresh CaCl2 (say 0.2mM) to give a final FREE [Ca2+] of 1.9 nM which should remain buffered during your experiment but is sufficient to activate the BK. Av alternative is to use Perforated Patch clamp instead of whole cell patch clamp in which the membrane is not ruptured put punctured with with hundreds of tiny holes by an ionophore like Gramacidin or amphotericin B. This allows the patching of whole cell currents but slows the exchange of cytosol with pipette solution.

For nicotinics you can include 0.9 mM EGTA in your patch pipette and 0.4mM CaCl2. The major source of the Ca2+ observed in your nicotinic whole cell current is going to come from the extracellular solution (where it will be about 2mM). The EGTA is not going to stop this, but it will buffer any large increases in intracellular Ca2+ due Ca2+ flux though the nicotinic channels.
wanderer36


Posted 1/18/2008 8:25:01 AM

...something else, if EGTA is add to one solution, is normal that the solution become more alkaline? Because EGTA is one acid, so one can think should be the contrary...

Thanks.


frasermoss


Posted 1/18/2008 8:19:10 PM
You should have 10mM HEPES or an equivalent in both your patch pipette solution and extracellular solution to buffer any potential pH changes
wanderer36


Posted 1/21/2008 11:14:19 AM

...yes, the solution have 10 mM HEPES, and when I add EGTA, it becomes more alkaline; of course I can adjust it with some acid, is just that I dont understand why it become more alkaline if EGTA is one acid. ... ... ...

guy


Posted 1/30/2008 6:25:46 PM
Hi ,
Regarding EGTA concentration in BHK cell experiments, what is the highest concentration which you tried?
wanderer36


Posted 1/31/2008 8:58:38 AM
...in the pipette we used 0.5mM CaCl2 and 5mM EGTA; in the external solution we have 2mM CaCl2. Could be good or something should be chage to get better results?

frasermoss


Posted 1/31/2008 6:06:19 PM
I'm sorry, can you repost that last message...it was hard to follow with what i believe are a few typo's
Thanks
wanderer36


Posted 1/31/2008 8:01:58 PM
Kein problem.

...I said, in the solution for the micropipette for whole cell recordings, we put 0,6mM of calcium and 5mM EGTA, and in the external solution (ACSF) we used 2mM calcium. Could be good or something should be chage to get better results?

xiaoren


Posted 1/6/2009 8:19:53 AM


EGTA is a chelating agent useful for the determination/buffering of calcium in the presence of magnesium. If it contains in pipette with fluo-3/4-AM, would it affect intracellular calcium concentration induced by calcium wave,etc. Thanks!
Bluejay


Posted 1/6/2009 9:18:08 AM
One would expect EGTA to reduce calcium changes in the cell since it would chelate/bind and in a way, buffer the calcium levels. Dyes such as fluo and Fura were based on the EGTA strucuture. Theoretically you could calculate free calcium based on the Kd of EGTA and fluo (from Molecular Probes/invitrogen handbook). It may be easier just to prepare a standard curve using the anticipated intracellular fluo (non-AM form) and EGTA and varying the calcium.
There should be literature references detailing the experiment you suggested. You may want to look at the journal "Cell Calcium" published by Elsevier.
xiaoren


Posted 1/6/2009 10:00:41 AM
Thanks for your important recommendation!
andy123


Posted 9/21/2010 5:31:13 AM

It is useful for making buffer solutions that resemble the environment inside living cells where calcium ions are usually at least a thousandfold less concentrated than magnesium.

SPT


Posted 10/25/2010 11:17:35 AM
Can anyone provide insight into the optimal form of EGTA to use for whole cell patch clamping from neurons? We had been using the sodium salt, but it occurred to us that this might increase the intracellular Na+ concentration to non-physiological levels; is is better to use the potassium salt or free form?  Thanks for any ideas...
handsomejohanna


Posted 3/11/2013 3:57:18 AM
EGTA (ethylene glycol tetraacetic acid) is a polyamino carboxylic acid, a chelating agent that is related to the better known EDTA, but with a much higher affinity for calcium than for magnesium ions. It is useful for making buffer solutions that resemble the environment inside living cells[1] where calcium ions are usually at least a thousandfold less concentrated than magnesium.
Tran


Posted 10/28/2013 7:23:35 PM
 Some people use Cs-BAPTA in pipette as higher  affinity to calcium more than magnesium. What is the formula to calculate intracellular calcium concentration (for example: 100 nM Ca2+) based on  stock solution, 10mM Cs-BAPTA, 10 mM CaCl2  and 10 mM Cs-BAPTA and what software to calculate that? How can we know that?

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