Split cells using Trypsin and count them to obtain an accurate cell count. Centrifuge the cells and resuspend the cell pellet in starvation media, ensuring equal cell numbers across all treatment and control wells.
▶ 02:48
2
Set up transwell migration chambers
Add starvation media with epidermal growth factor (chemoattractant) to the lower chambers and starvation media with cell division inhibitor to the upper chambers. Seed a fixed number of prepared cells into each upper chamber.
▶ 04:39
3
Incubate cells to allow migration
Incubate the transwell plate at 37°C with 5% CO₂ for 24 hours to allow cancer cells to migrate through the porous membrane toward the chemoattractant.
▶ 05:53
4
Clean and remove non-migrated cells
Use cotton swabs to carefully clean the upper membrane surface of all cells that did not migrate through the pores. Remove the media from the upper chamber.
▶ 06:16
5
Fix cells with paraformaldehyde
Transfer each transwell to a well containing 4% paraformaldehyde fixative and incubate for 30 minutes to fix the migrated cells on the lower membrane.
▶ 07:23
6
Stain fixed cells with methylene blue
Add methylene blue stain to wells containing the transwells, ensuring the membrane is fully covered with stain. Incubate for 20 minutes to color the migrated cells.
▶ 07:59
7
Count migrated cells and analyze results
Rinse the stained membranes and observe under microscope. Capture 30 images (fields) per treatment, count cells in each field, calculate averages, and compare migration rates between treated and control samples.