Lab 8: Scratch Migration Assay using MDA-MB-231 Breast Cancer Cells
In this study, we will use phase contrast microscopy to observe migration of
MDA-MB-231 breast cancer cells. These cells have been grown on a thin layer of gelatin
in wells of a 4-well chamber slide, in the presence of fetal bovine serum. When exposed
to the growth factors in serum, the MDA-MB-231 cells form protrusions and migrate. In
this case, we will serum-starve the cells overnight, then pre-incubate two wells with the
Src inhibitor PP1 before treating one well with serum-free media and the other with
media containing serum to promote migration. We will then scratch the cell layer in each
well with the tip of a needle, and then observe migration as cells move into the scratched
area. After observing migration over time (about 1.5-2h), we will remove the media, fix
the cells, mount them with mounting media, and quantify the amount of cell migration in
each well using phase contrast microscopy and a micrometer.
Scratch migration assay
Procedure
1. The cells will be serum-starved overnight to remove growth factors, and will then
be pre-incubated with the Src inhibitor PP1 or with the solvent DMSO (in the
appropriate treatment groups) for at least one hour, then treated with serum (in the
appropriate groups) for another hour. Treatment groups: serum free media +
DMSO, serum free media + PP1, media with serum + DMSO, media with serum
+ PP1.
2. Using an 18-gauge needle, make a vertical scratch (lengthwise) in the confluent
cell layer. Check the scratched areas using phase contrast microscopy to be sure
that you have scratched a cell monolayer and that the scratched area is clear of
cells.
3. Carefully remove and discard the culture media, depositing the media into a
container with bleach.
4. Wash 1 time in 1X PBS to remove cellular debris. Remove PBS and place into
container with bleach.
5. Add fresh serum-free media, adding the same treatments that were used initially:
serum free media, serum free media + PP1, media with serum, media with serum
+ PP1.
6. Monitor migration every half hour to ensure that they don’t close completely (or
you will not be able to quantify differences in migration). The goal is to get the
scratch in the serum-treated well with no PP1 to close almost completely. When
this occurs, stop the assay.
7. Remove media, wash cells with 1 X PBS. Discard both media and PBS into
container with bleach.
8. Add 500 ml formalin to each well of the chamber slide to fix the cells. Fix for 10
min.
9. Remove the formalin, and discard into marked container in the hood.
10. Wash the cells with two, 5 min changes of PBS. Place discarded PBS into the
formalin waste container.
11. Carefully remove the plastic, top part of the slide chamber, saving the microscope
slide with the cells. You will also need to carefully remove the rubber gasket
surrounding the bottom of each well using a flat spatula or razor blade.
12. Place 40 ml of 0.1% (1 mg/ml) phenylenediamine and DAPI in 50% glycerol (in
H2O) on top of the cells in each well of the slide and place a cover slip on top of
the drop. Be careful NOT to create bubbles between the coverslip and the
cells.
13. Blot the top of the coverslip with a kimwipe to remove excess mounting media.
14. Seal the cover slip to the slide with nail polish. If you do not observe the cells
immediately, store the slide in your labeled slide box in the refrigerator for future
observation.
15. You may observe the cells and quantify cell
migration using the small scopes in the lab,
using objective of 10x-20x. To quantify
migration, use the ocular micrometer (located
in the eyepiece) to measure the number of
units in the remaining gap. You can then
convert the units to micrometers using this
conversion:
10X, 10 mm/unit
20X, 20 mm/unit
16. To ensure greater accuracy, measure the number of units at multiple locations for
each scratch. You may then use this data to determine the average gap size and
the standard deviation for each well. Graph your results, and determine whether
there are significant differences among the different treatment groups.
17. You may also observe the cells and quantify cell migration using the large
fluorescence scopes in the microscope room. To quantify migration using this
scope, you save each picture as a JPEG 2000; when you do so, make sure that you
select the appropriate objective. You can then determine length of the gap by
clicking the mouse on either side of the gap, and the software calculates the
distance in micrometers for you. You can then make a series of measurements
that can be exported to Excel for further analysis—specifically, to determine the
mean and standard deviations, graph your results, and determine whether there are
significant differences among the different treatment groups.