Do someone know if the addition of EGTA to the bath solution can affect or change the intracellular calcium? I ask because EGTA is suppose to be cell impermeant, or not?
Hello. How much EGTA? If it is mM range then, yes, it can. If you reduce Cao, eventualy this will affect Cai homeostasis. this of course will very much depend on cell types. If they are excitable the effect of adding EGTA will be larger. I would recommend you to load cells with fura 2 or anyother dyes and monitor the effect of EGTA to check the safe time window you would have for your experiments.
...yes, there is 2mM EGTA and the cells are neurons. It will be good if I can see if the intracellular Ca++ change. Can I know it with fura 2? When the cells are loaded with fura and calcium imaging is performed, how can I know if the intracellular calcium change when there is EGTA (or something else) in the perfusion? (I´m beginner in calcium imaging)
What do you mean?
If you have a cell loaded with Fura 2 and a a fluorimetric system, you just have to monitor control fluorescence and then perfuse EGTA. To estimate Ca concentration changes you will need to apply a simple formula that relates F380, F360 and Kd for fura-2.
For a video tutorial of how to perform calcium Imaging using Fura-2 follow this link
...I don´t have any cell loaded with Fura. I´m just performing whole cell Patch Clamp and would like to know if when EGTA is added to the bath, can affect the intracellular calcium and if someone know some paper that talk or explain about it (I´m not performing calcium imaging).
Have a look to this paper. They made quite simple Ca measurements. I would also recommend you to search Pubmed for the effect of EGTA in resting Ca2+. Good luck.Pflugers Arch. 2000 Jan;439(3):304-14.
Ulate et al
Extracellular ATP regulates exocytosis
by inhibiting multiple Ca2+ channel types in bovine chromaffin cells
...I also have the option to use calcium imaging (even I´m not performing it for now). So far I understand, in loaded cells with fura-2 you can see the calcium coming into the cell. Is there possible the contrary, to see if intracellular calcium decrease?
ps... I keep to other topic too until I can clarify better.
Yes, of course, you can see Ca decreases as well.
...sorry if I lloks a little lost (or a big)
...so, if the cells are loaded with fura, you can see the changes on calcium currents for calcim coming into the cell, so calcium currents are induce and then measure with the calcium imaging; it means two tings: calcium currents have to be induce in some way and the changes or decrease that we can see are only in this calcium coming into the cell.
Now, if the loaded cells are just in the chamber and I want to see if there are changes in the intracellular calcium just perfusing without Ca++, for example, is there possible? If yes, how it looks during the "recordings"?
mmm, yes, you are a bit lost! ;)
Well, I strongly recommend you to read about Ca2+ homeostasis. Ca currents are not the only way to control intracellular Ca concentration. You have, Ca stores, mitocondria, Ca pumps and exchangers, endogenous buffers, etc. As a result, fura 2 florescence will change. (fure-2 is a fluorescent probe that changes it fluorescence emission depending on intracelluar Ca concentration.
I think you need to deeply study these topics before anything else.
Good luck (and don't worry, we have all been at that stage, just take time!)