Hi there, I'm currently working on a project where i need to design an experiment but as i have little to no experience with this level of experimental design and no face to face help i am really struggling and so any advice would be really greatly appreciated! I am currently designing an experiment to observe the affects of calcium dyshomeostasis on neuronal dysfunction in alzheimers disease and have opted to design an in vitro model using cultured Tg2576 cells.
Firstly, do you think it will be enough to create different cell culture environments with different amounts of 59mM KCl addition to cause calcium overload over 40 minutes (e.g control with no calcium, culture 1 with 1 addition of KCl, culture 2 with 2 additions of KCl etc.). I have struggled to find a method of causing calcium dyshomeostasis in vitro.
Secondly, once the cultures have been exposed to the calcium, i aim to measure the affects of this on the neurons' membrane potentials. I aim to measure this via the whole-cell patch clamp method. Would this be an effective way to do this? Do i need to use a specific microelectord to do this and what buffer solution should i use both in the culture and in the microelectrode?
I think you may have trouble keeping your neurons alive and well for current clamp if you are depolarizing with extracellular K+ for so long. If you are trying to get Ca2+ inside the cell you could try a Ca2+ ionophore like ionomycin (I think there are others as well) but that also tends to make cells unhealthy after a bit of time.
Jacob, in my humble opinion, you cannot keep your cells healthy under such
conditions. 40 min membrane depolarization with 60 mM KCl to induce the Ca2+ influx is too rough and will simply kill the cell.
I tend to agree with Dr Hays comment above to try to use ionomycin or A23187 but at very low concentration. Ideally you need to combine your patch-clamp rig with confocal microscope and preload your neurons with Fluo 3 to see how much of Ca you are really getting into the cell. Once you establish how much Ca ions you "allow" into the cell and not killing it immediately and still maintaining it resting potential, you may figure out how many Ca2+ you need.
Another approach would be, I think, is to apply your 60 mM KCl, let Ca2+ into the cell, do your recording and then replace external solution with low KCl. Something like that.
Cassandra L Hays , Evgeny Petrov thankyou both for your advice! I will consider your points further and attempt to adjust my plan to make it more viable using your recommendations!
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