Tracking Axonal Transport of Organelles in Motor Neurons Using a Microfluidic Device
Transcrição
Dispose of all medium from the proximal compartment of the MFC. Pick up a single spinal cord explant with a pipette in a total volume of four microliters and inject it as close as possible to the cave. Draw out any excess liquid from the proximal well via the lateral outlets. Repeat the previous step two more times, and make sure that the explants are embedded in the proximal channel. Slowly, add 150 microliters of SCEX medium to the proximal well.
One day after plating, replace the SCEX medium in the proximal compartment, and add rich SCEX medium to the distal compartment, maintaining a volume gradient of at least 15 microliters per well between the distal and proximal wells. After four to six days, the axons should cross to the distal compartment and be ready for axonal transport imaging.
To label the mitochondria and acidic compartments, prepare fresh SCEX medium with 100-nanomolar Mitotracker deep red FM and 100 nanomolar LysoTracker red, and add it to the microfluidic chambers. Incubate them for 30 to 60 minutes at 37 degrees Celsius, then wash three times with warm SCEX medium. Proceed with live imaging of axonal transport, acquiring a 100-time lapse image series at 3-second intervals with a total of 5 minutes per movie.
