I am trying to establish a fluorescent glutamate release. This assay uses NADP, Calcium chloride and Glutamate dehydrogenase enzyme. Glutamate dehydrogenase acts on released glutamate( which is released on depolarizing synaptosomes by KCl, 4-AP) and NADPH is produced which gives a florescence signal at 340 nm (emission wavelength) and 460 nm (emission wavelength) I am preparing Synaptosomes by Percoll gradient method. I am not able to deploarize synaptosomes using potassium chloride. I have tried increasing NADP, Glutamate dehydrogenase but nothing seems to give a signal with KCl. I am not able to find a reason for no effect by KCl.