Cell Migration Assay Using Transwell Chambers

Protocol Steps

1. 1
   Cells are washed in cold PBS,...

   Cells are washed in cold PBS, detached with 5mM EDTA, centrifuged at 1200 g for 7 min and resuspended in serum-free medium.

2. 2
   Transwell chambers (Corning Costar) are prepared...

   Transwell chambers (Corning Costar) are prepared filling with 600 μl of complete medium the lower chamber.

3. 3
   The desired amount of cells is...

   The desired amount of cells is plated in 100 μl of serum-free medium in the upper insert (8.0 μm pore size) of each Transwell chamber and incubated at 37°C.

4. 4
   After 24 hours at 37°C, fill...

   After 24 hours at 37°C, fill with 600 μl of 5mM EDTA an empty well beside each used Transwell chamber.

5. 5
   Eliminate DMEM from the upper insert...

   Eliminate DMEM from the upper insert and move it inside an EDTA well.

6. 6
   Collect DMEM from the lower chamber...

   Collect DMEM from the lower chamber and put some EDTA inside, to detach cells possibly fallen from the upper insert.

7. 7
   Detach cells migrated on the underside...

   Detach cells migrated on the underside of the upper insert using a plastic scraper and collecting them in the EDTA well.

8. 8
   Mix in one tube cells collected...

   Mix in one tube cells collected from the lower chamber and scraped from the insert and centrifuge at 1200 g for 7 min, to count the migrated cells.

Materials & Reagents

Citation