Corning Transwell Cell Migration Assay Ultimate Guide

Cell migration and invasion play key roles in many normal biological processes as well as in cancer and other diseases. Corning Transwell permeable support migration assays are an essential research tool for understanding and manipulating these processes.

In a Transwell permeable support migration assay, a membrane separates upper and lower chambers, and researchers can test cells' ability to migrate toward an attractant by passing through pores in the membrane (migration assay) or by digesting a barrier that's used to coat the membrane (invasion assay).

Enabling Scientific Breakthroughs with Transwell Assay

Transwell permeable support cell migration and invasion assays facilitate pivotal research into phenomena like cancer metastasis, immune responses, cell communication, cell chemotaxis, and tissue healing.

For example, scientists are using Transwell permeable support migration assays to develop better systems for bone tissue engineering, advancing the quest for laboratory-produced implants that could be used to replace damaged or diseased bone in patients. Researchers have also used Transwell permeable support cell migration assays to identify more potent chemoattractants for bone marrow stromal cells and to test the ability of engineered scaffolds to recruit bone marrow mesenchymal stem cells.

Other researchers are applying Transwell permeable support cell invasion assays to make strides in cancer biology. For example, the authors of a 2020 study used Transwell permeable support assays to investigate whether antibodies that inhibit matrix metalloproteinase enzyme MMP-2 can block oral cancer cell invasion. They found that the antibodies greatly decreased the tumor cells' ability to migrate and invade through a barrier, pointing the way to future research.

Materials Checklist for Transwell Assays

Here are the materials you will need to prepare for a typical Transwell permeable support experiment:

  • Transwell permeable support cell culture inserts (come pre-assembled in a plate)
  • Corning Matrigel® matrix or other extracellular matrix (for invasion assays)
  • Syringes (for invasion assays)
  • Cells or cancer spheroids whose migration or invasion will be tested
  • Attractant (such as cells, tissues, or chemoattractant media)
  • Culture media appropriate for your assay and cells
  • 0.2 μm filter unit (e.g., Corning 431222)
  • Cotton swabs
  • Sterile forceps
  • Positive displacement pipet
  • Corning PBS (e.g., Corning 21-040-CM) for washing inserts
  • Suitable stain for microscopy or other imaging method (e.g., crystal violet, Diff-Quik, or hematoxylin and eosin) and staining/imaging supplies
  • Plate reader, microscope, flow cytometer, or other quantification device appropriate for your assay
  • Other materials specific to your assay

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Step-by-Step Procedure for Transwell Permeable Support Assays

Here is a general procedure for easy assay execution that can be varied for your specific needs:

  1. Using aseptic technique, culture the cells or spheroids whose migration or invasion you will test.
  2. If performing an invasion assay, follow manufacturer's instructions to reconstitute or prepare extracellular matrix.
  3. If performing an invasion assay, use a sterile syringe or pipet to apply extracellular matrix to each insert (except uncoated control inserts), minimizing contact with the side walls. Follow manufacturer's instructions to allow extracellular matrix to form a gel. Alternatively, use pre-coated and pre-assembled options, Corning BioCoat® invasion chambers or Transwell permeable supports.
  4. Prepare cell suspensions. Seed suspensions of cells or spheroids into each upper chamber at desired cell density and volume.
  5. Place desired attractants in lower chambers in appropriate media.
  6. Incubate for the desired timespan.
  7. Collect or fix, stain, and image invaded or migrated cells to quantify migration or invasion.
    • For nonadherent cells, remove the Transwell inserts and collect cells in the lower companion plate. Then, count or stain and image the cells using your preferred method.
    • For adherent cells, one inexpensive method is to first remove non-invaded or non-migrated cells by gently but firmly wiping the upper surface of the insert with a cotton swab moistened with PBS. Then, fix and stain cells that have migrated to the underside of the membrane.
    • A potentially higher-throughput method for adherent cells is to dissociate cells from the underside of the membrane and use an appropriate quantification method (e.g., Calcein AM).
    • Quantification can be performed by microscopy with image capture, microscopy with direct cell counting, flow cytometry, counting with a hemacytometer, or use of a plate reader. If using microscopy, be sure to include enough fields near the center and edges of the insert.
    • For invasion assays, calculate percent invasion by dividing the mean number of invaded cells by the mean number of cells migrated through control, uncoated membranes and multiplying by 100.

Troubleshooting Cell Migration and Invasion Assays

Migration and invasion assays are complex, and optimization of multiple aspects of a Transwell permeable support migration assay is often required. Here are some considerations:

  • Choose the proper pore size for the cells you're studying. Look for a pore size that migrating cells can squeeze through but not fall through — and one that's as relevant as possible to the biological system you're studying.
  • The measured percent migration can be inaccurate at low or high seeding densities because of problems like oversaturation of pores, oversaturation of assay signal, or conversely, low sample size. Run a titration using a known chemoattractant to determine the optimal cell density for your experiment.
  • Also, try titrating the chemoattractant concentration and the migration time for your experiment. Consider testing different chemoattractants and invasion barriers.
  • Be sure to include proper controls. This will help you detect when something has gone wrong and decide how to fix the problem. For example, in invasion assays, including a low-invasion cell line as a control can help you confirm that the extracellular matrix barrier is effective at stopping the movement of non- or low-invasive cells. Including a control well with the experimental cells and no extracellular matrix coating will allow you to calculate percent invasion. Including a well with no chemoattractant and a well with a known chemoattractant as negative and positive controls, respectively, can help you optimize and troubleshoot migration assays.
  • Be sure the cells are not damaged before seeding them in the cell culture inserts. Some harvesting methods (such as the use of trypsin) can damage receptors and make cells less sensitive to chemoattractant. If cells are not migrating adequately, try serum starving them for 24 to 48 hours to increase their sensitivity to the chemoattractant.

Corning's Cell Migration and Invasion Assay Products

Corning products can support a wide variety of invasion and migration assays including Transwell permeable supports with different pore sizes, formats, membrane materials, growth areas, and coatings.

Corning offers reliable tools and protocols for cancer metastasis research, including leading products for intravasation and extravasation assays. Get in touch today to learn more.