Endosome recycling preparation

Fluorescent styryl dyes such as FMl-43 have been used to approximate neurotransmitter release by measuring rates of ex-ocytosis (16, 72, 73). These dyes reversibly label endosomal membranes and can be taken up into intracellular synaptic vesicles during endocytosis in systems in which vesicle recycling takes place. Typically, tissue is incubated in the fluorescent dye and then stimulated to promote vesicle cycling and therefore uptake of the dye. The preparation then is washed in fresh buffer to remove dye that remained extracellular. Using fluorescent microscopy, vesicle dynamics can be tracked. Neurotransmitter release is estimated from the rate of destaining (because of exocytosis) usually during stimulation. 

The combined methods of preparing an endosome-enriched fraction labeled with acridinium-transferrin and specific immunoadsorption of unlabeled Rabll-positive recycling endosomes provide the basis of our in vitro fusion assay. Using this assay, transport of the physiological marker transferrin from labeled donor endosomes to immunoisolated acceptor recycling endosomes can be followed by measuring arrival of acridinium-transferrin to beads-bound endosomes as detected by lashlight-luminescence in a luminometer. 

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