TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

The overall goal of this procedure is to investigate neurotransmitter vesicle dynamics in neuroblastoma cells using pH sensitive probes and total internal reflection fluorescence microscopy or turf earth. This is accomplished by first plating cells onto glass cover slips and transecting them with genetically encoded pH sensitive fluorescent probes of vesicle fusion and recycling, which are selectively targeted to synaptic vesicles. Protein expression is expected within 24 to 48 hours.

The second step is to record vesicle dynamics by total internal reflection microscopy. This step is particularly critical for the success of the experiment and a step-by-step description on how to achieve turf configuration is provided.Next. Once the correct turf configuration is reached, the sequential images can be automatically recorded by the software in basal conditions or stimulated conditions.