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Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell    

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Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, usually a tissue-culture-treated microporous membrane, which is positioned between two compartments that mimic two different sets of microenvironments for cell survival/growth. Cells on one side of the membrane, when sensing chemoattractants placed on the other side of the compartment that diffuses through the membrane, can migrate through the pores in the membrane towards the source of the chemoattractants. Cells that migrate across the membrane can be quantified by fixing and counting. Human breast epithelial adenocarcinoma MD-231 cells grow relatively fast and are metastatic. The MB-231 cell line is used here to describe the procedures of an in vitro cell migration assay using the transwell apparatus.

Materials and Reagents

  1. Human MDA-MB-231 cell (ATCC, catalog number: HTB-26 ™)
  2. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
  3. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  4. Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
  5. Trypsin inhibitor (soybean) (Life Technologies, Invitrogen™, catalog number: 17075-029 )
  6. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
  7. Collagen I (Sigma-Aldrich, catalog number: C7661 ) or Fibronectin (BD Biosciences, catalog number: 354008 )
  8. Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
  9. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  10. Crystal violet (Sigma-Aldrich, catalog number: C3886 )
  11. TCC-formulated Leibovitz's L-15 Medium (ATCC, catalog number: 30-2008 ™)


  1. Corning® Transwell® polycarbonate membrane inserts (Sigma-Aldrich, catalog number: CLS3421 ) or Millicell Cell Culture Inserts (EMD Millipore, catalog number: PI8P01250 )
  2. Cotton swabs
  3. Cell culture incubator: 37 °C and 5% CO2


  1. Carry MB-231 cells in DMEM with 10% FBS (use L-15 medium if needed).
  2. Wash cells twice with 1x PBS and trypsinize.
  3. Add 0.5 mg/ml Trypsin inhibitor in PBS to inactivate an equal volume of Trypsin. Aspirate cells by pipetting up and down gently (Note: It is important to break down into individual cells as much as possible).
  4. Gently spin down the cells. Wash cells two times with DMEM containing 0.5% FBS to remove trace amounts of trypsin and inhitibor. Resuspend the cells in DMEM with 0.5% FBS and count.
  5. Prepare the transwell compartments, 24-well format, with 8 μm pore size insert:
    1. To the lower compartment, add 2.6 ml of DMEM with 0.5% FBS containing 40 μg ml-1 Collagen I.
    2. Add the transwell insert to the well by merging the bottom of the insert into the medium in the lower compartment.
    Note: Ensure that no air bubbles are trapped between the insert membrane and the medium.
  6. To the upper compartment, gently add 1 x 105 cells from step 4.
  7. Incubate the cells in the transwell plate at 37 °C and 5% CO2 for 2.5 h. This allows cells to migrate toward the underside of the insert filter.
  8. After 2.5 h, carefully take the insert out. Cells that do not migrate through the pores and therefore remain on the upper side of the filter membrane need to be gently removed with a cotton swab. Gently wipe the upper side of the filter membrane with a cotton swab to remove the cell debris. We recommend that use each clean cotton swab for one wipe only, in one direction and do not swipe in back-and-forth movement. The cotton swab can be slightly moisturized with ddH2O as needed but be sure to remove any excess water. Several wipes may be needed to completely remove any cell debris on the membrane.
  9. Fix the cells on the lower side of the insert filter quickly with 5% glutaraldehyde for 10 min.
  10. Next, stain cells on the lower side of the insert filter with 1% crystal violet in 2% ethanol for 20 min.
  11. Remove excess crystal violet by quickly merging the insert in ddH2O for 3 to 4 sec. Drain excess water from the side of the insert using a cotton swab. Dry the insert membrane.
  12. Count the number of cells on the lower side of the filter under a microscope. Randomly choose different views and take average counting.
  13. The same experimental procedure should be performed for control groups without chemoattractants. Each migration condition should be tested with replicates.


This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Katoh et al. (2006) and Nakamizo et al. (2005). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee  [see Chen et al. (2009)].


  1. Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
  2. Katoh, H., Hiramoto, K. and Negishi, M. (2006). Activation of Rac1 by RhoG regulates cell migration. J Cell Sci 119(Pt 1): 56-65.
  3. Nakamizo, A., Marini, F., Amano, T., Khan, A., Studeny, M., Gumin, J., Chen, J., Hentschel, S., Vecil, G., Dembinski, J., Andreeff, M. and Lang, F. F. (2005). Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 65(8): 3307-3318.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chen, Y. (2012). Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell. Bio-protocol 2(4): e99. DOI: 10.21769/BioProtoc.99.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Young-Sang Kim
PuKyong National University
Hello, I performed transwell migration assay with HT29 cells.
when I take pictures, for counting cells, I cannot distinguish the cells and pores(see the picture).
So, I need help for staining and washing and how to take a picture using microscope.
Other papaers with same experiment, their figures are very clear, when i read papers.
please Help me.
1/28/2016 11:55:46 PM Reply
Judy Tang

There is nearly no cell in your picture. Maybe lengthen the time of culture or put more cells may help.

3/24/2020 6:01:30 PM Reply

xl w
shanghai chempartner
Hi, is the membrane is permeable to collagen? If so, the concentration of collagen will equilibrate between the up and low compartment. How collagen can still work as chemattractants?
Thank you!
2/19/2014 4:04:46 AM Reply
Yanling Chen
The Scripps Research Institute

This experimental setup allows gradient of chemoattractant be established, which is the "driving force" for migration. We don't know how long it takes to "equilibrate" if that would happen eventually, time is also a determining factor here, I think.

2/21/2014 8:29:57 AM Reply

Lisha Chen
Hi, I would like to know why the collagen I were added in the medium in the lower compartment. I saw some experimental procedures say the uupper chambers should be pre-coated with rat collagen type I.What's the function of the rat tail collagen I in this assay? Thank you!
7/2/2013 10:56:38 PM Reply
Yanling Chen
The Scripps Research Institute

Collagen is added as a chemoattractant; you may use other chemoattractants of your choice.

7/19/2013 7:03:50 AM Reply

Lisha Chen

Thank you!I have another question. Is the 2% ethanol in crystal violet solution necessary? If I use the solution that crystal violet dissovled in ddH2O, can it work?

8/13/2013 1:18:07 AM Reply

Yanling Chen
The Scripps Research Institute

I guess that should work too as long as you can get it dissolved well.

8/13/2013 7:30:49 AM Reply

It would be really helpful if you could be more specific about the volume in which we need to dispense the cell in each insert . Just cell number does not help. Also what will be the volume of agonist in the lower chamber and cell in upper chamber in a 96 well plate?
10/16/2012 4:01:16 AM Reply
Hi there,

I have few questions regarding this essay. First, MDA231 cells in my hand didn't migrate to the centre of the insert. After migration (24 h), I saw lot of cells at the rim of the insert. I tried to change the way i added cells, volume of media and cell numbers in the insert. But still none of these worked. I always used 10 % FBS as the chemoattractant (I used comoplete media for this). My first try was ok. Cells migrated evenly through the membrane. But after that I could not get the same result.
Could you please suggest me what would be the problem here?

Many thanks
10/9/2012 8:20:21 PM Reply
Yanling Chen
The Scripps Research Institute

1. Please make sure that the bottom of the insert, when immersed in medium, has no small bubbles trapped underneath (especially the middle area). The microporous membrane should be soaked evenly in medium.
2. Please add the volumes of media/cell according to manufacturer's manual, which keeps the inside/outside media at certain levels.
3. Avoid swirling medium when adding cells to the insert.
4. The transwell plate should be kept steady when incubated, reduce moving it around or shaking it.
5. For your experiments, you may also way to try different cell seeding densities, and/or a shorter migration time.
Hope these can help.

10/17/2012 9:34:08 PM Reply

Will MDA-MB-231 cells migrate/invade with FBS and not EGF? I have read several papers that imply this cell line will not invade/migrate without it. Thanks!
1/24/2012 6:14:35 AM Reply
Yanling Chen
The Scripps Research Institute

In the author's hands, EGF has never been required for successful migration assay using MB231 cells. Some researchers even carry out this assay using medium without either FBS or EGF. Please see reference: PLoS One. 2010 Dec 30;5(12):e15940.

1/28/2012 6:07:03 PM Reply

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