I am trying to measure calcium levels in hippocampus rat slices, specifically in CA1 layer, using membrane-permeable Fura-2. I am using control slices incubated without Fura-2 but with DMSO, the dissolvent in which I resuspend Fura-2. I can observe fluorescence in my slices incubated with Fura-2 but the problem is that I observe fluorescence in the control slices too. I know that autofluorescence could be the reason, but when I add KCl 1M I can observe and strong increase both in slices incubated with Fura-2 and control slices. Do you know what can be happening? Is it possible that DMSO produce fluorescence? Thank you ;)
Yikes! 1M KCl will depolarize your cells to the point where excitotoxicity is almost a guarantee - since intracellular proteins, NADPH, etc. are autofluorescent, you might be seeing their release en masse through compromised membranes.
Thank you Jonny. We were aware that 1M is a extremely high concentration, but we just wanted to see a positive control to check that Fura is working; because of that. we always add KCl at the end of the experiment. However, I have not thought about excitoxicity and membrane compromis, so, we will try today with KCl 100mM, or have you got any idea about the optimal concentration? Again, thank you very much for your contribution
Hi Lucas,
there is little chance for this to be the problem but are you doing auto-exposure when you change from your loaded slice versus your control? Ideally you would get the exposure parameters from your slice that has been treated as to prevent saturation and then use the same parameters in the control slice. If you auto-expose the control slice differently then you can not compare them.
Regarding KCl concentration 10mM is a common concentration used in similar studies; I agree than 1M is totally overkill and might be part of your problem.
Hope this helps,
Ezequiel
To my knowledge DMSO does not fluoresce, and if it did it would be at extremely small wavelengths because the promotion of localized pi electrons requires a great deal more energy than the aromatic electrons usually associated with fluorescence.
I've used 25mM KCl and my cells were certainly not happy with that, but since you're just looking for a control it might be ok. 10mM might be a good idea as Ezequiel suggested. Since you are just looking for a positive control, there are many things you could try if that doesn't work: if you have a perfusion system that can do it, pulse the high [K+] solution instead of just bathing longterm; give a shot of glutamate, bicuculline, gabazine, etc.; use a stimulating electrode to depolarize your cells temporarily; the list goes on.
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