I am working on a protocol to measure calcium flux by synaptic signaling in live mouse neuron culture. I am using Fluo-4 AM for my stain, which I have seen others use, but it is not showing any flux or change in signal that I'd expect to see with movement of synaptic vesicles. Has anyone had success with this stain or suggestions for an alternative calcium stain I could use? Alternatively, any other ways to measure synaptic communication? Any help is greatly appreciated.
Thank you!
Fluo-4 AM is commonly used for monitoring calcium flux in cells. If you are not observing the expected signal, it may be due to a number of factors, including the presence of calcium buffering agents, the presence of other fluorescent dyes, or problems with the excitation or detection systems. You may want to try using a different calcium stain, such as Fura-2 AM, to see if that resolves the issue.
Another alternative for measuring synaptic communication is to use electrophysiological techniques such as patch clamping or multi-electrode arrays. These methods directly measure the electrical activity of neurons and can provide information about the release and reuptake of neurotransmitters, as well as the flow of ions across the cell membrane. Additionally, you may also want to consider using optical imaging techniques such as two-photon microscopy to visualize changes in synaptic vesicles in real-time.
Sudarshan Bhattacharjee Thank you for your response! I'll try to move forward with your suggestions.
How old are the cultures? Are they healthy? If neurons are too young, they have not formed complete synapses yet, and the overall signal might be too weak to be detected. Which buffer do you use for the assay (HBSS, HBTS)? Does it include calcium? I use Calcium6 dye from Molecular Devices and it works well in FLIPR mode, and I used Fluo-4-AM in single-cell calcium imaging. Have you tried a positive control for calcium influx such as ionomycin? If ionomycin works well, you should easily detect calcium influx, suggesting that your dye works - maybe the compounds you use are not so potent (concentrations can be adjusted)? Good luck, I hope this helps!
Gabriella Saro The neurons look completely healthy and seem to be alive (I can see some movement in the cytoplasm during imaging). The staining is done in HBSS, and I have tried most of your suggestions in some form - but this makes me wonder if I have been staining them too early. I will try some later timepoints. Thank you!
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