To begin fibronectin coating, select 12 inserts and 2 24-well plates. Place the inserts into one well plate, creating two rows of six. Prepare one microgram per milliliter fibronectin solution by mixing 52 microliters of fibronectin stock solution and 1.2 milliliters of PBS in a 1.5 milliliter tube.
Distribute 100 microliters of the fibronectin solution into each insert. Incubate the inserts in the well plate at 37 degrees Celsius for three hours. After incubation, remove the excess fibronectin solution from each insert.
Wash the inserts twice by adding 100 microliters of sterile distilled water to each insert. Remove the water in the same order as it was added to ensure consistent wash time between inserts. Now wash the inserts with 100 microliters of cell culture media, and then remove the media from the inserts.
Next, seed 200 microliters of A431 cell suspension into the inserts. For negative control, pipette 200 microliters of cell culture media into the inserts. Using a permanent marker, draw a line dividing the second well plate into two columns that are three wells wide.
Separate each column into rows. Label each region in the grid with relevant parameters. Add one milliliter of cell culture media to each well.
Transfer the inserts from the preparation plate to their appropriate location in the labeled experiment plate and incubate at 37 degrees Celsius for 12 hours.
