Practical Guidance for Optimizing Cell Migration and Invasion Assays

Cell migration, a term used to describe the directed movement of cells in response to a stimulus, is critical for normal physiological processes such as embryogenesis, wound healing, and immunity. However, cell migration also has a central role in various disease states, including tumor cell invasion into healthy host tissue during cancer metastasis. This article explains the basic principles of cell migration and invasion assays and shares tips for generating reliable results.

Migration vs. invasion assay fundamentals

The most widely accepted cell migration technique is the Boyden chamber assay, first reported in 1962 for studying leukocyte chemotaxis.¹ The method involves seeding cells into a hollow plastic chamber, sealed at one end with a porous membrane, which is suspended over a larger well containing medium and/or chemoattractants. After allowing sufficient time for migratory cells to move through the pores, any cells remaining on the apical (top) side of the membrane are gently removed with a cotton swab. Migratory cells on the basolateral side of the membrane are then fixed and stained prior to imaging and counting. Invasive cells may be similarly measured by coating the membrane with an extracellular matrix (ECM) gel, such as Corning^® Matrigel^® matrix. The principles of cell migration and invasion assays are shown in Figure 1.

Figure 1. Principles of cell migration and invasion assays. Cells are seeded into porous inserts, which are suspended in medium containing the relevant chemoattractant. Following incubation, cells on the apical side of the membrane are gently removed with a cotton swab, and migrated/invaded cells are fixed and stained before imaging and counting. Invasion assays involve coating the membrane with an ECM, whereas this step is omitted for migration assays.

Selecting the right pore size

Pore size is a key consideration when setting up a cell migration or invasion assay and should be carefully matched to the type of cells you intend to use. Typical pore size options include 3 μm, which is appropriate for studying leukocyte or lymphocyte migration; 5 µm, which is suitable for working with monocytes, macrophages, and cancer cells such as NIH-3T3 and MDA-MB 231; and 8 μm, which supports optimal migration for most epithelial and fibroblast cells. Critically, large pore sizes should be avoided for smaller cell types, while small pore sizes should not be so small that they prohibit migratory cells from reaching the chemoattractant. Researchers are advised to consult the literature when determining which pore size to use for specific migratory and invasive cell types.

Key optimization variables

Besides pore size, there are several other important variables that should be optimized when running cell migration/invasion assays. These include the following:

• Cell seeding density—this should be high enough to prevent inconsistent counting, but not so high that it risks oversaturating the pores in the membrane. A suggested plate layout for optimizing the cell seeding density is shown in Figure 2.
• Chemoattractant concentration—for a well-studied chemoattractant, such as fetal bovine serum (FBS), a concentration of 10% serum is a good starting point. If there are no previously published data on the interaction being investigated, performing several different serial dilutions of the chemoattractant is recommended.
• Incubation time—16–24 hours is usually sufficient, but some cell type/chemoattractant combinations may require longer.
• Fixation method—4% paraformaldehyde and 70% ethanol are commonly used.
• Type of stain—popular stains include 0.5% crystal violet (whole cell staining) and 1 µM DAPI (nuclear staining). When combining alcohol fixation with crystal violet staining, cells should be imaged and counted within a few days to avoid alcohol distortion of morphology.

Figure 2. Example of a 24-well plate layout for optimizing seeding density. Replicate testing is important to generate reliable results.

Cell migration and invasion assay controls

Controls are essential for monitoring assay performance. Examples of negative controls for cell migration and invasion assays include wells with no chemoattractant, wells seeded with a non-migratory cell line (e.g., MCF-7), and wells lacking an extracellular matrix coating (cell invasion assays only). Positive controls vary depending on the assay, but typically consist of wells containing a known chemoattractant.

Practical tips for success

To help you avoid common pitfalls for cell migration and invasion assays, here are our five top tips: 

• Always suspend cells in serum-free media to avoid any contamination that could limit their sensitivity to the chemoattractant.
• Prior to seeding, carefully break up any clumps with a smaller bore pipettor to avoid aggregation of cells in the middle or edges of the insert.
• Ensure that cell culture inserts are seated correctly to allow for even migration/invasion.
• Make sure the lid of the plate can close properly to prevent evaporation during incubation.
• Store stained samples at 4 °C in molecular-grade water to prevent the cells from drying out.

MilliporeSigma (the Life Science business of Merck KGaA, Darmstadt, Germany) offers an extensive selection of products for cell migration and invasion assays, including Millicell^® Cell Culture Inserts and Plates. To learn more, visit https://www.sigmaaldrich.com/GB/en/products/labware/cell-culture-and-cryogenics/millicell-cell-culture-inserts-and-plates

Reference

1. Boyden S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med. 1962;115(3):453-466. doi:10.1084/jem.115.3.453