To begin, rapidly thaw a frozen stock of SH-SY5Y cells in FBS. Supplemented with 10%DMSO and dilute tenfold with pre-warmed media. Then centrifuge the cells and remove the supernatant.
Re-suspend the cell pellet in five milliliters of pre-warmed media and seed cells in a T25 flask. To prepare cover slips coated with PDL apply 600 microliters of 50 micrograms per milliliter PDL solution to each well of sterile chambered cover slips. The volume of PDL added to each well varies based on well size.
Incubate the cover slips set room temperature for one hour. After incubation, remove the PDL solution and rinse the cover slip thrice with 1.8 milliliters of distilled water. Allow the coated chamber to air dry for two hours.
Once the SH-SY5Y cells reach 80 to 90%confluency, dissociate them by adding trypsin solution. Neutralize the trypsin by adding a pre-warmed DMEM medium. Centrifuge the cell suspension and re-suspend the pellet in DMEM.
Count the cells on an automated cell counter. Next, seed the cells at a density of 1.5 times 10 to the fourth cells per square centimeter onto the PDL coated chambered cover glass. On day one and day three, replace the medium with DMEM supplemented with either five or 2%FBS respectively, 1%antibiotic antimycotic, and either 10 micromolar RA or 95%ethanol of the same additive volume to serve as the vehicle control for differentiation.
Prepare a PKMO stock in pre-warmed Phenyl red free DMEM supplemented with two or 10%FBS dependent on differentiation state, 1%antibiotic antimycotic, and 20 millimolar hepes. On day six, rinse the cells twice with imaging media and incubate cells with the PKMO solution at 37 degrees celsius and 5%carbon dioxide for 30 minutes. After staining, rinse the cells thrice with pre-warmed imaging media.
For the final wash, incubate the cells for 30 minutes at 37 degrees celsius, and 5%carbon dioxide. After incubation, replace the final wash with fresh pre-warmed imaging media containing 20 millimolar hepes.
