Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

In This Article

Abstract

Source: Tsuruta, F., et al. Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes. J. Vis. Exp. (2017).

This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles' movement.

Protocol

1. Imaging Vesicle Motility

Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for late endosomes, LAMP-EGFP for lysosomes, and EGFP-LC3 for autophagosomes.
NOTE: These plasmids are available on request from tsuruta.fuminori.fn@u.tsukuba.ac.jp. Typically, 3 -5 days in vitro (Division or DIV) neurons can be transfected with these plasmids using transfection reagents.
Mix 4.0 µg of plasmids with 200 µL of serum-free medium by pipetting. In a separate tube, dilute 8 µL of transfection reagent (see Table of Materials

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Results

LAMP-EGFP vesicle motility analysis; fluorescence imaging, graph, bar chart; position vs. time, mobility distance measurement.
Figure 1: Lysosome Motility in Neuronal Dendrites. (A) Mouse cortical neurons (4 DIV) were transfected with LAMP-EGFP plasmid for 2 days. This image shows the motility of LAMP-EGFP-containing vesicles in the dendrites. The inse.......