Stimulating Neuronal Cell Differentiation Using Gold Nanorods
Transcript
Grow the NG108-15 neuronal cells in 10 milliliters of cell culture medium in a T75 flask, subsequently incubate it at 37 degrees Celsius in humidified atmosphere. When the cells are 70% to 80% confluent, change the medium with warm, fresh medium.
After that, mechanically detach the cells by gently knocking the bottom of the confluent flask. Centrifuge a cell suspension for five minutes at 600 g and resuspend the cell pellet in 2 milliliters of warm cell differentiation medium. Then, seed the cells in a 96-well plate, with 200 microliters of cell differentiation medium.
Incubate the sample for one day at 37 degrees Celsius. The next day, add the gold nanorod solution and incubate it for an additional 24 hours. In this procedure, couple the laser with a single-mode optical fiber and terminate it with an FC connector. Measure the output laser power with a standard power meter.
On day 3 of nanorod incubation, fix the FC connector to the well, irradiate the sample, and the control at room temperature for 1 minute in a continuous wave at different laser powers. On day 5, remove the cell differentiation medium from the sample and fix it with 3.7% volume per volume formaldehyde solution for 10 minutes.
Then, permeabilize the cells with 0.1% volume per volume Triton X-100 for 20 minutes. Afterward, add 3% weight per volume BSA to the sample for 60 minutes to block the unreacted protein-binding sites. Label the sample with anti-beta-3-tubulin overnight at 4 degrees Celsius.
The next day, incubate the cells for 90 minutes in the dark, with an appropriate secondary antibody. Subsequently, label the cell nuclei with DAPI for 10 minutes, then, image the sample with epifluorescence or confocal microscopy, using at least a 20x objective. Choose the microscope filters according to the secondary antibody, and select the DAPI filter to visualize the cell nuclei.
