Luminescence-Based Assay to Assess Neurotoxicity of Test Compounds on Neurons
Transkript
To coat the plate, add 30 microliters of poly-L-lysine into each well of a 384-well plate, and incubate the plate at room temperature for 1 hour. Wash it twice with PBS, and let it dry for approximately 30 minutes.
Add 30 microliters of laminin to each well, then, incubate the plate at 37 degrees Celsius for 2 hours. Repeat the washes with PBS, and proceed with plating the cells. Add 20,000 single-cell neurospheres in 25 microliters of differentiation media, to each well of the 384-well plate, and incubate the plate at 37 degrees Celsius for 5 days.
High-precision pipetting skills are required for plating the exact number of neurospheres that are disaggregated into single cells. Therefore, differentiating the neurospheres into neurons, before plating is recommended to achieve more reproducible results.
After the incubation, add 5 microliters of test compound to each well at 6 times the desired concentration, and incubate the cells for another 24 hours.
To perform the cell viability assay, add 30 microliters of luminescent reagent to each well, and leave the plate on a shaker for 2 minutes. Centrifuge the plate at 300 to 400 times g for 30 seconds. Then, incubate the plate at room temperature for 10 minutes, protecting it from light. After the incubation, measure the luminescence on a microplate reader.
