Begin by plating SHSY 5Y cells in glass bottom cell dishes at a density of 15 by 10 rays to the power four cells per milliliter. Treat the cells with all-trans retinoic acid in 1%FBS containing culture medium for five days, followed by treatment with brain-derived neurotrophic factor for two days. Then, wash the cells with sterile PBS.
Treat the cells for 72 hours with 10 to 7 molar RA or isoform agonists using equal parts of MEM and F12 medium combined with 1%FBS. Replace the medium with fresh 1%FBS containing culture medium mixed with 20 nano molar TMRM for 45 minutes. Place the cells in an incubator connected to a laser scanning confocal microscope set at 37 degrees Celsius.
Capture the images using a 63x oil immersion apochromatic objective. Set the image size to 512 by 512 pixels with a pinhole aperture of one area unit. Collect a time series of 15 frames from five different visual fields in each cell plate and a Z stack of eight equidistant Z-planes.
The resulting LSM file should contain time, position, and Z-stack data.
