Improve Research Reproducibility A Bio-protocol resource
bpdetail_icon_lock
Preprint

Cell migration assay

YC Yu-Chia Chen
AS Aussie Suzuki

Last updated date: Jun 9, 2026 Views: 3 Forks: 0

original An abbreviated version of this protocol was published in eLife in May, 2025
Inhibition of p38-MK2 pathway enhances the efficacy of microtubule inhibitors in breast cancer cells
download pdf Download PDF
ask Ask a question
cite How to cite
nfavorite Favorite
  1. Starve Cal51 cells by incubating in DMEM-0 (0% serum) overnight (more than 20 hr).
  2. Trypsinize the starved Cal51 cells and resuspend cells with DMEM-10 (10% serum). The serum used here is to neutralize Trypsin to minimize the cytotoxicity.
  3. Count the cell number using a hemocytometer under microscope. 
  4. Pellet the cells to the bottom of a 1.5-ml microcentrifuge tube by centrifugation (800 g, RT, 2 min).
  5. Aspirate the media and resuspend cells with an appropriate amount of DMEM-0. Pipette 30 times to fully resuspend the cells. The cell density should be 3x10^4 cells / 50 μl. 
  6. Transfer 50 μl of cell suspension (3x10^4 cells) to a new 1.5-ml microcentrifuge tube. The number of tubes depends on the number of tested conditions.
  7. Prepare 50 μl of DMEM-0 with drugs at 2-fold concentration. If the final concentration of the drug is 1 μΜ, then it should be 2 μM in this step. 
  8. Add 50 μl of drug containing DMEM-0 into 50 μl of cell suspension (3x10^4 cells). Pipette 30 times to mix. This will be loaded in the upper chamber of the insert. 
  9. Prepare 500 μl of DMEM-10 with drug in a new 1.5-ml microcentrifuge tube. The drug concentration should be 1x. This will be added in the outer well. 
  10. Add 500 μl of DMEM-10 with 1x drug in the outer well. 
  11. Use tweezers to hold the insert and put in the well containing 500 μl of DMEM-10 with 1x drug. 
  12. Add 100 μl of cell suspension in DMEM-0 with 1x drug into the upper chamber of the insert. In this step, both inside and outside the insert has the same concentration of drug. But the media inside contains no serum whereas media outside contains 10% serum to generate the serum concentration gradient. 
  13. Put the cell culture plate in 37°C incubator and incubate the cells under drug treatment for 16 hr (overnight) to allow cells to migrate. The actual incubation time may need to be optimized depending on the drug and cell type. 
  14. Warm up 4% PFA at 37°C water bath. 
  15. Add 500 μl of 37°C 4% PFA to a well in a 24-well cell culture plate. 
  16. Transfer the cell-containing insert into PFA-containing well by tweezers.
  17. Aspirate the media in the upper chamber of the insert by suction.
  18. Add 100 μl of 37°C 4% PFA to the upper chamber of the insert by P200 pipette.
  19. Fix the cells in PFA in 37°C incubator for 10 min. 
  20. Aspirate PFA both inside and outside the insert by suction. 
  21. Wash the insert with PBS twice (outer well: 500 μl; upper chamber of insert: 100 μl).
  22. Permeabilize cells with methanol at RT for 2 min. 
  23. Wash the insert with PBS twice (outer well: 500 μl; upper chamber of insert: 100 μl).
  24. Stain the cells in 1% crystal violet (Electron Microscopy Sciences, cat. no. 26105-01) at RT for 2 min. 
  25. Wash the insert with PBS three times (outer well: 500 μl; upper chamber of insert: 100 μl).
  26. Use the cotton swab to remove the cells in upper (interior) side of the insert so that only cells successfully migrating to the lower side of the chamber will be left. 
Copyright: © 2026 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Chen, Y and Suzuki, A(2026). Cell migration assay. Bio-protocol Preprint. bio-protocol.org/prep2954.
  2. Chen, Y., Takada, M., Nagornyuk, A., Yu, M., Yamada, H., Nagashima, T., Ohtsuka, M., DeLuca, J. G., Markus, S. M., Takaku, M. and Suzuki, A.(2025). Inhibition of p38-MK2 pathway enhances the efficacy of microtubule inhibitors in breast cancer cells. eLife. DOI: 10.7554/eLife.104859

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A
This protocol preprint was submitted via the "Request a Protocol" track.