I am trying to load calcium Green Tm AM inside the neurons. As a first step, I would like to ensure the dye permeates the cell membrane. I am using a 5x and 10x objective to quantify the background fluorescence, then, I inject 200ul of a solution containing the dye and get some pictures post-injection. There is a difference from the background image, but I don’t have a cellular resolution. Would anyone have any suggestions on how I could approach this? I am not sure, for example, whether I can image the dye (it could also be fluo-4, I have a number of those, and I am trying to establish a protocol to check their levels of cell permeability) post PFA fixation using a confocal microscope.
Thank you very much for your time!
The AM esters are trapped inside the cells once they get in because of hydrolysis of the esters. If you wash the cells after incubating with the dyes to remove any external dye, you should be able to observe increased fluorescence in the cytoplasm of the living cells when calcium release occurs.
Thanks a lot, Adam, I understand the principle of the technique, but I am having problems loading the dyes. Do you have experience with AM dye loading in live tissue?
I don't have such experience. I think these things are more commonly used with cells in culture. The problem with tissues may be that the extracellular fluid may contain esterase enzymes that cut off the ester groups before the compounds can get into the cells. You may be able to fix this by preincubating the tissue with an esterase inhibitor, if you can find one that can't get into the cells.
- Badges
- Science topic