Cell migration is the directed movement of cells in response to a chemical (e.g., chemoattractant) or mechanical stimulus. It is an important property of cells, critical for cell development and normal processes such as wound healing. However, it is also involved in disease states such as inflammation or tumor cell migration and invasion.¹ Migration and invasion assays have long been utilized in cell biology for the study of tumor metastasis, with the most common assays based on the Boyden assay system. This system was first used in 1962 by Dr. Boyden where he demonstrated the use of Millipore® porous membrane for chemotaxis.²
Today, migration assays can be carried out using Millicell® hanging cell culture inserts based on this Boyden assay system, while invasion assays require coating of the membrane with an ECM gel, such as Matrigel®, and then seeding the cells on it. This coating acts as a barrier for cells to penetrate during invasion. Inserts with different pore sizes are available, dependent on the size of the cells to be used.
The Boyden Chamber system uses a hollow plastic chamber and is sealed at one end with a porous membrane. This chamber is then suspended over a larger well which may contain medium and/or chemoattractants. Cells are washed in serum free media to ensure removal of all serum before being added to the apical (top) side of the membrane in the inserts. Media containing the relevant chemoattractant (e.g., Fetal Bovine Serum, various cytokines etc.) is added to the wells below the inserts (along with a negative control where no chemoattractant is in the media), and the cells are left in a 37°C CO₂ incubator for the desired amount of time.
Following this incubation, non-migrated cells are gently removed from the apical side of the membrane and the migrated cells on the basolateral side of the membrane can be quantified using cell staining and imaging.
Figure 1.Invasion assay principle. Cells are seeded in ECM-coated inserts on apical side of the membrane, while media containing the relevant chemoattractant is added to the cell culture plate. Plates are left for the required time (e.g., 24 hours) to allow for invasion of cells to the basolateral side of the membrane. Non-invaded cells are removed with a cotton swab, and invaded cells are fixed and stained before imaging and counting. For migration assays, simply omit the ECM-coating step.
The pore size of your insert depends on the type of cells that are to be used in your assay. It’s important that too large of a pore size isn’t used for smaller cell types, but the pores can’t be so small that migratory cells cannot fit through even in the presence of the chemoattractant. Table 1 shows membrane pore size recommendations for a selection of migratory and invasive cell types based on literature.
Millicell® hanging cell culture inserts have several options compatible with migration and invasion assays (shown in Table 2). All inserts are individually blister-packed and fit standard tissue culture plates.
Samples can be saved and re-stained if needed. The inserts can be stored at 4 °C in molecular-grade water.
Figure 2.Example of 24-well plate layout for optimizing seeding density for cell migration assays (or invasion assays if using Matrigel®-coated inserts).
Migration and invasion assays were performed as described in the previous section. Crystal violet staining was carried out following these 24-hour migration assays, for three cell lines (HT 1080, NIH 3T3, and MCF-7) with and without 10% FBS. This allowed for migrated cells to be imaged clearly, showing the level of migration for each cell line under each condition (images shown in Figure 3). HT 1080 cells are capable of invasion and migration, NIH 3T3 cells are capable of migration only under the conditions outlined in this experiment, while MCF-7 cells are incapable of migrating through the pores towards the chemoattractant.
Figure 3.Crystal violet staining of migrated cells (A – HT1080, B – NIH 3T3, C – MCF-7) on basolateral side of membrane after 24 hours, with and without 10% FBS and Matrigel® (200 µg/mL), at different seeding densities. Representative images are shown from one field of view, from one insert replicate at each seeding density. HT 1080 and NIH 3T3 cell lines can migrate through the pores towards the chemoattractant, while MCF-7 cells cannot. Only the HT 1080 cell line can invade through the Matrigel®-coated membrane.
In addition to crystal violet staining, cells were also stained with DAPI to allow for imaging and counting using a fluorescent microscope (shown in Figure 4). Imaging was completed using a 10x objective with the DAPI filter. Once the images were acquired, migrated/invaded cells could be counted in four fields of view for every insert. DAPI is a nuclear stain, which allows for easier counting of migratory/invaded cells, especially when there are large numbers of cells present in the higher seeding densities.
Figure 4.DAPI staining of migrated cells (A – HT1080, B – NIH 3T3, C – MCF-7) on basolateral side of membrane after 24 hours, with and without 10% FBS and Matrigel® (200 µg/mL), at different seeding densities. Representative images are shown from one field of view, from one insert replicate at each seeding density. HT 1080 and NIH 3T3 cell lines can migrate through the pores towards the chemoattractant, while MCF-7 cells cannot. Only the HT 1080 cell line can invade through the Matrigel®-coated membrane.
The objective of this experiment was to optimize seeding density for a migration and invasion assays using 10% FBS as a chemoattractant for three different cell lines, following 24 hours of incubation. The number of cells that migrated or invaded in each insert was calculated by averaging the number of cells across four fields of view following DAPI staining and imaging. Graphical representations of the results are shown in Figure 5. These results allowed us to determine that the optimal seeding density in this experiment was 25,000 cells per insert for both migration and invasion assays. There was a good separation of cell counts between inserts that contained the chemoattractant (+FBS) and the negative control (-FBS), with 25,000 cells per insert seeding density showing the best signal-to-noise ratio for the two migratory cell lines (5:1 and 6:1 for HT 1080 and NIH 3T3 respectively). Corresponding controls were tested for each cell line to compare both with and without FBS conditions (data not shown). Appropriate seeding density is dependent on the specific conditions to be tested by the user (e.g., time of migration, the cell line used, chemoattractant used, etc.)
Figure 5.Quantification of migrated cells on the basolateral side of membranes after 24 hours, with and without 10% FBS and Matrigel® (200 µg/mL), at different seeding densities (showing the average of cells counted from four fields of view, from three insert replicates). HT 1080 and NIH 3T3 cell lines can migrate through the pores towards the chemoattractant, while MCF-7 cells cannot. Only the HT 1080 cell line can invade through the Matrigel®-coated membrane.
Figure 6.Crystal violet staining of Millicell® inserts. A. Too much pressure was placed on the Millicell® insert when removing it, indenting the membrane and causing the insert not to be in the same focal plane when imaging. B. Crystal violet staining of Millicell® inserts using ethanol as a fixative agent. When using ethanol as a fixative agent, samples should be images as soon as possible to avoid ethanol distortion of cell morphology.
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