Talk:Calcium Green

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Cell permeable Ca2+ fluorophores

See also Calcium

Question:

Can I use the cell permeable ester derivative of Calcium green-1 (Calcium Green™-1, AM, cell permeant) to measure INTRACELLULAR Ca concentrations of INTACT cells? How do I a calibrate?

Answer:

Calcium green-1 is the type of Calcium Green for the lowest Ca concentration. Since we are talking intracellular Ca Concentration calibration of Ca measurements in the chamber volume (NOT what YOU want to do!) would probable have to be performed by preparing calibration buffers --> too low concentrations for just titrating in Ca. Of course the application I always have in mind is using PERMEABILIZED cells or ISOLATED MITOCHOBDRIA and adjusting the medium in the measuring chamber to simulate the INTRACELLULAR medium. This is what we do in 95% of our applications (no matter which method: O2, TPP, pH). For Ca there is the special problem that keeping the chamber volume similar to intracellular medium usually involves buffering away all the Ca introduced by the preparation and all the chemical to protect the mitochondria. This is were the different approaches in the literature (e.g. using a low Ca buffering capacity in the initial phase of the experiment, then "out-titrating" this buffer with Ca pulses) come into play. Another application is to have INTACT cells and look at comparable high EXTRACELLULAR Ca concentrations - again looking at the Ca concentration in the bulk of the medium, NOT IN the sample. This should be rather simple due to the high Ca levels, but this Ca level will not change a lot by things happening inside the cells. In all cases so far you would use the water soluble form of Mg green (the hexapotassium salt).

However, if I understand correctly you propose to do something entirely different: I get that you want to measure INTRACELLULAR Ca concentrations in INTACT cells and this is why you bought the cell permeable form for Ca green-1 (the ester). So you would not be interested in the signal from the bulk of the medium but only from inside intact cells. While this is not what our fluorescence system is designed for, it does not mean that it would not work. Actually, if a measurement is possible at all (and I do not know if it is possible!) there are some benefits. My problem is that I know absolutely nothing about this cell permeable preparations, especially not if they are slightly water soluble and therefore how much of the fluorophore stays outside the cell. Just from looking at the Invitrogen page I gather that usually these dyes are measured when it is possible to look just at the cell(s), e.g. with fluorescence microscopy. Whether it is possible to detect the signal from inside the cell in the presence of the bulk medium around(that our sensor is also seeing) I do not know and I guess this will depend strongly on

1. The answer to the question I asked above: How much of the fluorphore is dissolved in the medium? That is how much of an (uninteresting and disturbing) background signal is generated by the medium outside?

2. Is the signal from inside the cells strong enough to be measured, considering that the detector is seeing mostly the medium outside?

The first step would be to look at the "measuring methods" applied in the literature using such cell permeable Ca-green derivatives:

• Fluorescence microscopy? -not very helpful for our task
• Flow cytometry: probable also not very helpful, because the measurement is still focused on a single cell at any given time
• Cuvette based measurements : very helpful : If it works in a cuvette in should also work in the O2k measuring chamber.

The good point (if this works at all) is of course that you do not have to care about Ca buffering- the intact cell is taking care about that.

Calibration: You can not calibrate by either by titration or by calcium buffers because you can not set a defined Ca2+ concentration INSIDE an INTACT cell. Please refer to the manual (and the supplied references) from the fluorophores producer.

I always prefer to test a system first without any biological sample to make sure the instrument system works well. However in this case I do not know how to do this. Even if the ester derivative of Calcium green -1 has some water solubility you would still have to set up a measurement system (calibration buffers) that is rather meaningless for the proper experiment. I guess one could start by comparing the fluorescence signals of a sample without fluorophore, a sample with fluorophore and sample with fluorophore plus tons of external Ca added. Since there is no way of lowering the Ca concentration in the cell, you have to trust the literature (and Invitrogen) that the fluorophore is designed in a way that ensures that at typical intracellular Ca concentrations the chelating part of the fluorophore is partially bound to Ca2+ and partially free. You only can detect a change in signal as long as you stay away from saturation or too low binding. Whether this condition is met will also depend on the ratio fluorophore/ cells you use.

Fasching Mario 15:43, 3 January 2014 (CET)