John J. Kim 08/28/2012 Conditioned Media (Migration) Assay Material: 1.

Chambers: Millipore: 24 Well Millicell Hanging Cell Culture Inserts 8,0 m PET CAT NO: PIEP12R48 2. Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane: Millipore; Cat# UFC901008 (cutoff: MW 10kD) 3. 24 well tissue culture plate and 6 cm cell culture dish. 4. 10% Serum RPMI 1640 Media 5. Serum-free RPMI 1640 Media

Protocol: 1. Seed equal number of cells (2 x105 5 x105 cells) for cell lines to compare in 6 cm cell culture dish. 2. Aspirate the 10% serum media. Add 5 ml of serum-free media to the 6 cm cell culture dish for starvation. Starve the cells for 48 hours for collecting conditioned media. Starve the cells for one night if there is already a collected frozen conditioned media and skip step 3 and 4. 3. After 48 hours, carefully collect the 5 ml of conditioned serum-free media. Spin down at 1000g for 10 min at the room temperature. 4. Carefully transfer the 5 ml of conditioned media to the centricone concentrator tube. Centrifuge at 3800g at 4 C until 1.25 ml left on the top of filter (~10 min). 5. Rehydrate the basement membrane layer of the cell culture inserts by adding 100 uL of warm, serum-free media to the inner compartment. Incubate at 37 C for at least 30 min. 6. Determine the final concentration of cells (0.1 1 x 106 cells/ml) for each well. Prepare a cell suspension containing 4x the final concentration (0.4 4 x 106) in serum-free media. 7. In a new eppendorf tube, add and mix 50 ul of 4x cell suspension and 150 ul of conditioned media. (For the frozen conditioned media, let the media thaw in ice and incubate for at least 5 min before mixing with cells to prevent cold shock.

John J. Kim 08/28/2012 8. Carefully remove the rehydration media (step 5) from the inserts without disturbing the basement membrane layer. 9. Add the 200 ul of cells and conditioned media to the inside of each inner compartment. 10. Add 700 ul of 10% serum media to the lower well of the 24 well plate. 11. Incubate for 6-24 hours at 37 C in 5% CO2. 12. Store the rest of conditioned media frozen at -80 C. 13. (Staining) Carefully aspirate the media from the inside of the insert. Wet the ends of 2-3 cotton-tipped swabs and gently swab the interior of the inserts to remove non-invasive cells. Take care not to puncture the polycarbonate membrane. Be sure to remove cells on the inside perimeter of the insert. 14. (Staining) Transfer the insert to a clean well containing 900 ul of Cell Stain Solution and incubate for 10 min at room temperature. 15. (Staining) Gently wash the stained inserts several times in a beaker of water. Allow the inserts to air dry.