To begin, take the Poly-D-Lysine pre-coated 384-well plate containing 25 microliters of laminin per well out of the refrigerator and equilibrate the plate to room temperature for about 30 minutes under the cell culture hood. Just before seeding, aspirate 15 microliters of coating solution from each well using an automated liquid handler or a 16-channel pipette. Then dispense 50 microliters of cell solution containing 300, 000 neurons per milliliter into each well.
Avoid filling cells in columns 1, 2, 23, and 24, and rows A, B, O, and P to minimize possible edge effects. Fill those unused wells with 80 microliters of phosphate-buffered saline. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide.
Prewarm an appropriate volume of complete maintenance medium at room temperature and away from light. Prepare a 1.5 times concentrated compound solution for all desired concentrations to be tested, using complete maintenance medium for dilutions. Using an automatic pipetting system, discard 40 microliters of medium per well from the neuron-containing plate, leaving 20 microliters of medium per well.
Then add 40 microliters of the 1.5 times concentrated compound solution per well to achieve the desired final concentration. Following the desired protocol, healthy donor, or LRRK2 G2019S mutation-carrying midbrain dopaminergic neurons were successfully cultured for six days, and treated with either dimethyl sulfoxide or a LRRK2 kinase inhibitor. The neurons were stained with nuclear stain and antibodies against alpha-synuclein, tyrosine hydroxylase, and MAP2.
