Visualizing Intracellular Transport of Cargo in Cultured Astrocytes

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