Migration assay

Detailed Protocol:

1. Add 700µl medium to be tested (e.g., conditioned medium, medium with inhibitor/cytokines/growth factors, etc.) in one well of 24-well companion plates (BD BioSciences)
2. Place the 8µM filter (BD Falcon cell culture insert, transparent PET membrane, Corning, Inc.) in the well.
3. Add tumor cells at a density of 5×10⁴ cells per filter in 300 μL medium.
4. Incubate at 37⁰C for 6 hours in the case of A549 cells. (Note: Optimize time of incubation according to cell lines)
5. After 5.5 hours, prepare the following material (for one 24 well plate)
   • A beaker with 1L 1X PBS
   • A beaker with 1L double distilled water
   • 24 well plate with 700µl/well Methanol
   • 24 well plate with 1:10 diluted 700µl/well Crystal Violet
   • Forceps
   • Wooden sticks with paper towels on them
   • Scalpels
     The following procedure can be done at once for 12 wells.
6. After 6 hours, take the filter with forceps, discard the medium, wash in a beaker with 1L 1X PBS, and dry the filter's inner side (NOT the outer membrane of the filter) with wooden sticks with paper towels on them.
7. After drying, immediately place the filter in 24 well plate with 700µl/well methanol according to its location.
8. Incubate at room temperature for 3 mins.
9. Place the filter in 24 well plate with 1:10 diluted 700 µl/well crystal violet for 10 mins.
10. After 10 mins, take the filter with forceps, discardthe medium, wash in a beaker with 1L double distilled water, and dry the filter's inner side (NOT the outer membrane of the filter) with wooden sticks with paper towels on them.
11. Place the filter on a clean bench with the outer membrane of the filter facing upward.
12. Cut the filters out with a sharp scalpel.
13. Place them in the slidesaccording to theirnumber (Note: (a) Place filtersin a way that migrated cells are to the downside to the slides (b) 3 filters can be placed on one slide).
14. Mount with Pertex (Medite GmbH) and cover with coverslips.
15. Scan slides with Nanozoomer 2.0HT digital slide scanner C9600 (Hamamatsu Photonics) and count the migrated cells per membrane using ImageJ software.