hi
the question I have done GFP plasmid transfection to the neuron cell this GFP plasmid is pH sensitive I need a way to make sure that the GFP plasmid is working by detecting membrane potential. for example, if the action potential is firing the GFP should give me a signal. it is supposed to give a signal ( green signal under the microscopy) when the pH change as a result of changing membrane potential
I can not add buffer with different pH because this GFP is inside the cell it is attached to the K+ channel.
I am thinking to use some pH indicator dye but not sure because it will be hard to do it at the same time to measure the fluorescent intensity of GFP and the dye. then I think doing the measurements separately and combining them together again this is will not tell me any thing about the membrane potential.
now I am thinking to use some ionophore that is specific to the K+ ion not sure.
@Malcolm Nobre
To check the functionality of your construct, cell lysates might work. Or try permeabilizing the cells with e.g. Streptolysin O.
Otherwise, can't you just evoke an action potential and record the response in the fluorescence channel, possibly by overexciting the cell and thus extending the time for recovery?
One way to do this would be to use a pH-sensitive dye or probe, such as fluorescein, to measure the changes in pH in real-time. Fluorescein is a widely used pH indicator dye that exhibits a change in fluorescence intensity in response to changes in pH. To use fluorescein, you would need to introduce it into the cells. This can be done through co-transfection or by adding the fluorescein to the culture media. Once the fluorescein is inside the cells, you can use a microscope equipped with a fluorescence detector to measure the changes in fluorescence intensity in response to action potentials.
Another option to consider is using a potassium-sensitive fluorescent probe, such as PBFI (potassium-binding benzofuran isophthalate). PBFI is a fluorescent dye that exhibits a change in fluorescence intensity in response to changes in potassium ion concentration. This could be a useful approach if you are interested in specifically measuring changes in potassium ion concentration in response to action potentials, as the GFP plasmid is attached to a potassium channel.
Regardless of which approach you choose, it is important to carefully control for any potential confounds, such as changes in temperature or changes in the extracellular pH, as these can also affect the fluorescence intensity of the probes.
Bertrand Cornu thank you so much for your answer but Do you think if I use fluorescein pH indicator or PBFI thats will give only their signal not GFP
I am afraid that will give only the signal for fluorescein pH indicator or PBFI
I need for example a pH indicator so I can measure both intensities for GFP along with the pH change
I will trigger the action potential for sure I will see the action potential peak that's for the control. for my experiment, I want to test after triggering the action potential the question am I able to see a difference in GFP intensity?
the action potential causes pH change and the gene in my plasmid GFP is a pH-sensitive so the hypises is when the cell fires action potinal the pH will change that will increase (stronger) or decrese ( weakens) the GFP signal
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