Counting Cells in Migration Assays with ImageJ

This application note describes a method to measure cell migration, using ImageJ, by counting the number of cells that have migrated into the Detection Zone in an Oris™ Cell Migration Assay. ImageJ is a freeware image analysis program developed at the National Institutes of Health (https://imagej.nih.gov/ij/).

Cell migration is critical for many physiological events, including embryonic development, wound healing, and the inflammatory response. Furthermore, aberrant motile behavior of cells contributes to pathological processes including tumor metastasis and arthritis (1).

The Oris™ Cell Migration Assay (Figure 1) uses a 96-well plate populated with silicone stoppers that exclude cells from the central Detection Zone of the well. After cells are seeded and allowed to adhere, the silicone stoppers are removed to reveal a 2mm diameter unseeded region in the center of each well, into which cells are permitted to migrate.

Figure 1. Schematic of Oris™ Cell Migration Assays

METHODS

MDA-MB-231 breast epithelial cells and HT-1080 fibrosarcoma cells were cultured on an Oris™ Cell Migration Assay – TriCoated plate having Tissue Culture Treated, Collagen I coated, or Fibronectin coated wells. After 16 hours, cells were fixed with 0.25% glutaraldehyde and cell nuclei were stained with 1:2000 DAPI (Pierce). Images were acquired using a 5X objective on a Zeiss Axiovert 200 inverted microscope equipped with a CCD camera.

Cell migration into the Detection Zone was measured by counting cell number using ImageJ analysis software (version 1.42l). First, the threshold was set for each grayscale image (Image–> Adjust–> Threshold). By selecting “Apply” in the threshold window, the thresholded image was converted to a binary image. Slightly overlapping nuclei were separated by performing a Watershed segmentation process (Process–> Binary–> Watershed).

Using the binary image, a circular region-of-interest (ROI) measuring 2mm in diameter (the same diameter as the tip of the stopper) was made using the menu command Edit–> Selection–> Specify. In the Specify window, “width” and “height” were set at 2mm, and the “oval” box was checked. The ROI was centered over the Detection Zone within each well. The number of nuclei contained in the ROI was quantified using the menu command Analyze–> Analyze particles. Values to define the minimum and maximum particle size were 100 and 1000 pixels2, respectively. “Show Masks” was selected to display a drawing of the detected objects. “Summary” and “Exclude on Edges” were checked for analysis.

The cell counts from the Summary Window (i.e., counts) were exported into Windows Excel for statistical analysis. The number of nuclei for each condition was averaged from 8 wells. Additional information regarding the use of ImageJ for particle analysis can be found at https://imagej.nih.gov/ij/docs/menus/analyze.html#ap).

RESULTS

In this application note, MDA-MB-231 and HT-1080 cell migration on three surfaces (Tissue Culture Treated, Collagen I, or Fibronectin; Oris™ Cell Migation Assay – TriCoated), was assessed by counting the number of cells in the Detection Zone using ImageJ. MDA-MB-231 cells exhibited varying degrees of migration into the Detection Zone dependent upon the surface coating of the well (Figure 2). Phase images of cells acquired immediately after stopper removal (migration control) and 16 hours after stopper removal demonstrate differences in cell migration depending upon whether cells were seeded on a Tissue Culture Treated surface, a Collagen I coated surface, or a Fibronectin coated surface (Figure 2A-D).

Using ImageJ, DAPI-labeled cells were counted by creating a 2mm circular region-of-interest (ROI) similar in size to the initial Detection Zone (Figure 2E-H). Performing the particle analysis function in ImageJ yielded drawings of detected objects that were counted within the circular ROI (Figure 2I-L). Differences in MDA-MB-231 cell migration into the Detection Zone (ROI) were highlighted by overlaying the particle analysis drawing on the original fluorescent image (Figure 2M-P).

Figure 2. ImageJ Analysis of Cell Migration by Counting Cell Number in the Detection Zone. (A-D) Phase images of migration control (A) and MDA-MB-231 cell migration on Tissue Culture Treated (B), Collage I (C), and Fibronectin (D) 16 hours after removal of stoppers from Oris™ Cell Migration Assay. (E-H), Fluorescent images of DAPI-labeled cells with a 2-mm circular ROI (red circle) defining the region for particle analysis. (I-L) Particle analysis drawings of objects in the ROI. (M-P) Merged images of DAPI and particle analysis drawings. Scale bar = 500 micrometers.

Figure 3 shows the average number of MDA-MB-231 (3A) and HT-1080 (3B) cells that migrated into the Detection Zone when seeded on Tissue Culture treated, Collagen I coated, and Fibronectin coated wells. Both MDA-MB-231 and HT-1080 cells exhibited the most robust migration on Collagen I. Furthermore, this method of analysis yielded statistical differences in the migration of model cell lines on all three plate coatings (i.e., MDA-MB-231 migration on Collagen I versus Fibronectin).

Figure 3. Quantitation of Cell Number using ImageJ. (A) MDA-MB-231 and (B) HT-1080 cell migration on three surfaces (Tissue Culture Treated, Collagen I, or Fibronectin). Data are represented as average cell number +\- SD from 8 wells for each condition. p<0.001; statistical difference between control vs. 16 hour migration on all surfaces; p<0.005; statistical difference in cell migration between surfaces (two-sample t-test).

CONCLUSIONS

This application note demonstrates a method to measure cell migration in the Oris™ Cell Migration Assay by the use of ImageJ analysis software for counting cells. This study, using ImageJ to quantify cell number in the Detection Zone, demonstrates that both MDA-MB-231 and HT-1080 cells exhibited statistically significant differences in migration when seeded on Tissue Culture Treated, Collagen I coated, and Fibronectin coated wells. Using the detailed analysis method described here, ImageJ can provide an accurate measure of cell migration when using the Oris™ Cell Migration Assay.

Cells on the Move: Do surface coatings influence cell migration?

Experiments show that surface coatings play an important role in cell movement

When performing cell migration experiments, a perennial question is: what surface coatings should be used to culture a particular cell type? Scientists working in Cancer Research, Wound Healing, or Drug Discovery utilize cell cultures to make important experiments and advance our understanding of biological mechanisms.  In particular, assays for cell migration enable characterization of conditions and substances that influence movement of cells.  For example, scientists using the OrisTM Cell Migration Assays successfully identified proteins, mRNA and antioxidants that inhibit migration of tumor cells. 

Each cell type requires specific conditions for optimal growth, so it is important to choose the right surface to culture each cell type.  For example, fibroblasts produce and inhabit the connective tissue of the body; thus we may hypothesize that surfaces and scaffolds that are rich in collagen and other fibers (components of connective tissue) are better suited for culturing fibroblasts.  This means that collagen-coated surfaces are a good choice when culturing fibroblasts for cell migration assays.  But does the choice of surface coating influence cell migration?

In new experiments, scientists at Platypus Technologies demonstrate that the choice of surface coating for cell culture may influence the results for cell migration experiments.  

Oris™ Cell Migration Assays use a 96-well plate with “stopper” barriers that create a central cell-free Detection Zone for cell migration experiments.  Removing the stoppers allows the cells to migrate into the Detection Zone at the center of each well:

Principle of OrisTM Cell Migration Assay: OrisTM stoppers are used to create a central cell free Detection Zone (red dotted circle).  Following 20 hours of incubation, the cells migrate into the detection zone.

In this experiment, the scientists used a cell line called HT1080, which are fibroblasts isolated from a malignant human tumor.  The cells were incubated on the OrisTM Cell Migration Assays that had wells coated with five different bioactive surfaces: Tissue Culture, Collagen I, Fibronectin, Poly-L-Lysine, and Basement Membrane Extract (BME).  Following cell attachment, the OrisTM stoppers were removed to permit cell migration into the detection zone.  The cells were then imaged following 24 hours of incubation without the OrisTM stoppers.  Representative images from this experiment are shown below:

Representative images of cells (HT1080) cultured in surfaces treated with various bioactive coatings.  OrisTM stoppers are used to create a central cell-free Detection Zone.  Following 24 hours of incubation, the cells migrate into the detection zone. HT1080 cells cultured in Collagen I surfaces migrate faster than when cultured in other surfaces.

Through visual inspection of these images, we observe that the cells cultured on Collagen I migrate deeper into the detection zone, compared to cells cultured on other bioactive surfaces.  There is no noticeable difference in the migration of the cells cultured on tissue culture, fibronectin, poly-l-lysine or basement membrane extract. 

Twelve (12) different wells from each plate were imaged prior and after cell migration, and the area of the cell-free zone was measured using ImageJ.  The difference in the area of the cell-free zone pre- and post- migration was used to calculate the percentage area closure of the assay.  These measurements are presented in the graph below, which show unequivocally that HT1080 cells exhibit higher migration rates in Collagen I than in any other surface coating. 

Quantitative Migration of Cells incubated on different bioactive surfaces.  These data indicate that the HT1080 cells incubated on Collagen I (Col I) exhibit higher rates of cell migration. 

In conclusion, the choice of surface coating has a strong effect on the cell migration of HT1080 cells.  Cells cultured in collagen I exhibit the fastest rate of cell migration, compared to cells cultured in other surfaces.  These results demonstrate that the choice of surface coatings plays an important role on the measured cell migration.

Learn more about OrisTM Cell Migration Assays: https://www.platypustech.com/cell-based-assays/oris-cell-migration

Learn more about 96-well plates coated with bioactive surfaces for cell culture: https://www.platypustech.com/cell-culture-solutions/microplates

External Links:

https://www.nature.com/articles/s41598-019-52480-3

https://www.thoughtco.com/how-and-why-cells-move-373377

New Surfaces for Cell Migration Assays

Platypus Technologies introduces new surfaces for Oris™ Cell Migration Assays: Poly-L-lysine and basement membrane extract.

Polylysine surfaces contain positively-charged amine groups that enhance attachment of negatively-charged proteins and cells. Polylysine surfaces are popular for culturing cell lines derived from nerve tissue (e.g. neurons, glial cells, fibroblasts, epithelial cells).

The basement membrane extract (BME) contains multiple extracellular matrix proteins, including laminin and collagen. Surfaces coated with BME support cell culture assays of epithelial cells, endothelial cells, muscle cells and stem cells.

Learn more about Cell Migration Assays: https://www.platypustech.com/cell-based-assays/oris-cell-migration

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