This article describes a method for monitoring intracellular cargo transport in mouse primary astrocytes using a fluorescent acidotropic probe. The technique involves time-lapse imaging under a confocal microscope to visualize the dynamics of labeled vesicles within the cells.
Begin with an adherent culture of mouse primary astrocytes on a coverslip within a culture dish.
Incubate with a fluorescent acidotropic probe that crosses intact cell membranes to enter the astrocytes.
The probe selectively accumulates inside acidic endolysosomal vesicles and fluorescently labels the vesicles to monitor their transport.
Wash to remove excess probe and add fresh medium.
Place the culture under a confocal microscope and focus on a single cell to perform time-lapse imaging.
Visualize the labeled cargo as fluorescent puncta distributed within the perinuclear region,
cytosol, and astrocytic processes.
Capture multiple focal planes along the Z-axis to visualize cargo throughout the three-dimensional cell volume.
Perform imaging at defined intervals to track the intracellular cargo transport at various depths within the astrocytes.
Analyze the cargo movement to distinguish between stationary cargo, cargo moving toward the cell periphery, and cargo moving toward the nucleus.
30 minutes before the imaging, dilute a suitable lysosomal labeling probe in 200 microliters of astrocyte culture medium to a working concentration of 1 micromolar, and label the astrocytes with the probe for 30 minutes at 37 degrees Celsius. Then wash the cells one time with warm astrocyte culture medium and replace the wash with imaging medium.
Immediately after the probe labeling, place the astrocyte culture container into the appropriate adapter on the microscope stage. And using epifluorescence light, locate the cells that express fluorescent proteins or probe. Use the digital camera to adjust the fluorescent sample illumination to visualize the selected cells, and adjust the focus and zoom into a single cell.
Then use the Zoom and definite focus functions to acquire a single C-stack time lapse series at a frequency of one frame every two seconds for time intervals ranging between 300 and 500 seconds. Save and export the time lapse images as Avi or TIFF stack files.
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