Single Cell Migration Assay Using Human Breast Cancer MDA-MB-231 Cell Line

Materials and Reagents

1. 24-well cell culture plate (Corning, Costar, catalog number: 9761146 )
2. 100 mm cell culture plate (Corning, catalog number: 08-772-22 )
3. 15 ml conical centrifuge tubes (Falcon, catalog number: 14-959-49D )
4. 100% CO₂ Tank
5. DMEM (Lonza, BioWhittaker, catalog number: BW12-604F )
6. Heat-inactivated FBS (Corning, catalog number: 35011CV )
7. 100x Antibiotic-Antimycotic (Gibco, catalog number: 15240-062 )
   Note: This is optional. We use this as extra assurance to prevent bacterial and fungal contamination in our cell culture. For some cell lines, this can hinder cell growth.
8. 100x Sodium pyruvate (Gibco, catalog number: 11360-070 )
9. Collagen I, 3 mg/ml (Gibco, catalog number: A1048301 )
10. Trypsin (Lonza, Trypsin-Versene, catalog number: BW17161E )
11. PBS (Lonza, BioWhittaker, catalog number: BW17516F )
12. MDA-MB-231 (ATCC, catalog number: CRM-HTB-26 )
13. DMEM S+ media (see Recipes)
14. Collagen (10 μg/ml) (see Recipes)
    

Equipment

1. Hemocytometer
2. Live-cell imaging environmental chamber (Tokai Hit live-cell environmental chamber)
3. Centrifuge (unrefrigerated) for 15 ml tubes (Sorvall, Legend RT, SO-LEGRT)
4. Cell incubator set to 37 °C and 5% CO₂
5. Waterbath or drybath set to 37 °C (Boekel Scientific, Small Water Bath, 290100)
6. Microscope with automatic stage control (Olympus IX71 Inverted Phase with Prior Scientific motorized stage) (Figure 1)
7. Camera for microscope (Hamamatsu)
8. 10x objective (Olympus)
   
   
   Figure 1. Example of a complete setup for cell tracking using Olympus IX71 inverted microscope, Prior mechanized stage, and Tokai Hit environmental chamber
   

Software

1. Software compatible with microscope for time-lapse image aquisition (Olympus, cellSens)
2. Fiji ImageJ (https://fiji.sc/)
3. Microsoft Excel
   

Procedure

1. Cell culture
   1. Warm up DMEM S+ media to 37 °C.
   2. Thaw MDA-MB-231 cells to 37 °C.
   3. Transfer 1 ml of thawed MDA-MB-231 cells to 3 ml of DMEM S+ media and centrifuge (with appropriate balance) for 3 min at 300 x g.
   4. Aspirate supernatant and resuspend cells in MDA-MB-231 cells in 10 ml of DMEM S+ and plate on 10 cm dish.
   5. Incubate cells at 37 °C and 5% CO₂ until 70% confluency (2-3 days).
      
      
2. Single cell migration assay
   1. Warm up DMEM S+ media, trypsin, and PBS to 37 °C.
   2. Coat 24-well plate with 10 μg/ml Collagen-I solution (100 μl) for 1 h at room temperature and wash with PBS (leave PBS in well to prevent drying).
   3. Aspirate media from MDA-MB-231 cell culture dish.
   4. Wash cells with PBS and aspirate PBS.
   5. Add 1 ml of trypsin and incubate in 37 °C and 5% CO₂ for 5 min.
   6. Pick up trypsinized cells with 3 ml of DMEM S+ and centrifuge for 3 min with appropriate balance at 300 x g.
   7. Aspirate supernatant and resuspend cells in 4 ml of DMEM S+.
   8. Take 10 μl of cell suspension and pipette into hemocytometer.
   9. Count cells.
   10. Aspirate PBS off collagen coated 24-well plate.
   11. Resuspend cell suspension again by inverting tube a few times or pipetting.
   12. Plate 20,000 cells into 24-well and allow to attach overnight at 37 °C and 5% CO₂.
   13. Allow live-cell imaging environmental chamber warm up on microscope stage with empty 24-well plate for at least 1 h to equilibrate microscope temperature.
   14. Replace empty plate with cell plated 24-well and allow to equilibrate for 1 h.
   15. Select multiple fields with 5-10 single cells (not touching other cells) and set time-lapse imaging for 2 h at 1 frame per minute using phase-contrast.
   16. Initiate time-lapse imaging and save files.
       

Data analysis

1. If fields were saved as individual images (i.e., 1 image per field per time point), import image sequence on Fiji to build a montage of all images from one field. If fields were saved as an individual file (i.e., 1 file per field), open directly with Fiji.
2. Play through the movie and identify cells that do not touch other cells or migrate off the field of view (try to identify 5-10 cells or more per field is possible). See Video 1 for example field of migrating cells.
   
   
   
   Video 1. Example video of cell migration from Figure 2
   
   
3. Once candidate cells have been determined, open Manual Tracking plugin on Fiji (Figure 2A, a window should open).
4. Click “add track” (Figure 2B).
5. Begin clicking on the centroid of cell (center of cell) and continue tracking until all 120 frames have been tracked (Figure 2C). Note that data will be added to a new window (Figure 2D).
6. Click “end track” and close manual tracking window (Figure 2E).
   
   
   Figure 2. Workflow for manual tracking using Fiji. A. Select Manual Tracking under Plugins. B. Click Add Track. C. Begin tracking a cell (continuously click on centroid of cell, for example, as illustrated by white arrow. Note that cells that are clumped together like those in the middle of this panel should not be used for tracking. D. Tracking information will be generated by ImageJ. E. Click End Track when tracking is done.
   
   
7. Save results window which contains X, Y coordinates of the cell centroid per frame.
8. Repeat Steps 3-7 until all cells have been quantified and proceed to next field.
9. Once all cells and fields have been quantified, open Excel and paste X,Y coordinates (Figure 3A).
10. Determine distance travelled from point to point by using the distance formula below where X₁ and Y₁ are the current track location and X₂ and Y₂ are the next track location. Find d for each tracked point (Figure 3B, there will be 121 tracked points, since there is no previous point to the first tracked spot, you will end up with 120 calculations for d).
    
    
    
    
    Figure 3. Example of data analysis using Excel. A. Copy over X and Y coordinates from cell tracking into Excel and line up side-by-side for each cell tracked. B. In separate section, use the distance formula to calculate distance travelled by cell from Point 1 to Point 2, Point 2 to Point 3, Point 3 to Point 4, and so on. Example of Excel function used is highlighted.
    
    
11. Add up all distance travelled per cell and divide by 120 (this is the duration of the movie in min and also the number of frames per field).
12. Repeat Steps 10 and 11 for all cells.
13. Summarize cell speed as desired and perform appropriate statistical analysis (t-test for 2 groups, ANOVA with post hoc for > 2 groups).
14. Plot summarized information using preferred graph (i.e., bar graph, box-whisker, scatter plot). An example of a box-whisker plot is shown in Figure 4.
    
    
    Figure 4. Example of speed results plotted as a box and whiskers chart using Excel. G1, G2, G3 are generic group names. The blue star represents the mean and the green bar represents the median of the data.
    

Recipes

1. DMEM S+ media
   500 ml DMEM
   50 ml FBS
   5 ml 100x Antibiotic-Antimycotic
   5 ml 100x Sodium Pyruvate
2. Collagen (10 μg/ml)
   10 ml DMEM
   166.6 μl Collagen I 3 mg/ml
   

Acknowledgments

This work was supported by a grant from the National Institute of Health (2R01CA108607) to PR. David Gau was supported by a National Science Foundation pre-doctoral fellowship (2012139050) and an NIH Cardiovascular Bioengineering pre-doctoral training grant (2T32HL076124 to SG).

Competing interests

The authors have no competing interests to report.

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