This protocol, the in vitro invasion assay, is a useful tool for quantitatively measuring a cells lines ability to migrate through a protein rich matrix and pass through a porous membrane, in a process similar to the early stages of cancer cell invasion. These cell lines can be genetically altered in order to over express a gene or to have reduced expression of a gene in order to determine if that gene plays a role in cell migration or cell invasion. Many aggressive cancers involve dramatic changes in cell behavior, including increased migration and invasion.
So this in vitro invasion assay can allow us to better understand the genes and other factors involved in this process. To begin this procedure, grow adherent mouse mammary tumor cells in a T25 flask at 37 degrees Celsius with 5%carbon dioxide and 100%humidity until they are 70%to 90%confluent for day one of the experiment. Retrieve the Boyden chamber inserts from storage and transfer them to a cell culture hood to warm to room temperature for 20 minutes.
Use three inserts to generate statistically valid data for each cell line for each experiment. And then add 500 microliters of the pre-warmed serum-free DMEM media to the inserts so that the porous membrane and gel are hydrated on both sides. Incubate at 37 degrees Celsius with 5%carbon dioxide and 100%humidity for at least two hours.
After this, prepare the cells by removing the growth media and rinsing the cells with five milliliters of HBSS. Remove the HBSS and add one milliliter of 0.25%trypsin EDTA solution. Incubate at 37 degrees Celsius for one to five minutes or until cells appear rounded or show signs of detachment.
Then gently tap on the flask to detach all the cells. Re-suspend the cells in five milliliters of DMEM with 10%FBS. And transfer the cell suspension to a sterile 15 milliliter centrifuge tube.
Centrifuge the cells and 1000 times g for five minutes to gently pellet the cells. Remove the media and re-suspend the pellet in five milliliters of serum-free DMEM. Repeat the centrifugation and re-suspending process two additional times for a total of three washes.
After this, thoroughly re-suspend the cells in the final five milliliters of serum-free DMEM and ensure there are no clumps of cells. Use a hemocytometer to determine the cell concentration making sure to only count viable cells. And dilute the cell suspension to 50, 000 cells per milliliter in five milliliters of serum-free DMEM.
Next, remove the dish with the inserts from the incubator, and gently aspirate the media from an insert. Lift the insert and aspirate the media from the well. Working quickly, add the chemoattractant to the lower chamber.
Place the insert into the well, and for a 24 well dish, add 500 microliters of the cell suspension to the insert. Ensure that there are no air bubbles present on either side of the membrane. Return the dish to the cell culture incubator for 22 hours.
After 22 hours, prepare a solution of 1%paraformaldehyde and one X PBS for fixation. In a clean 24 well cell culture dish, add one milliliter of the fixative to individual wells so there's one well for each insert. Then, prepare the staining solution of 0.1%crystal violet in a solution of PBS with 10%ethanol.
Add one milliliter of this staining solution to a clean well for each insert. Using forceps, remove each insert one at a time. Place a sterile cotton swab inside each insert and swab the upper side of the membrane to remove any unmigrated cells.
Repeat this process with a second cotton swab. Next, remove any remaining media from the inside of the insert and add 750 microliters of PBS to wash away any detached cells. Remove the PBS and repeat the wash with fresh PBS.
Then place the insert into a well containing fixative to fix the migrated cells on the underside of the membrane. Repeat this process for all inserts and let the inserts fix for 15 minutes at room temperature. After this, remove each insert one at a time from its well and wash it with 750 microliters of PBS.
Place the insert into a well containing staining solution and leave it for 15 minutes to stain all migrated and fixed cells. Then remove the inserts and dip them into a beaker containing distilled water until the water running off the insert is clear. Remove any excess water droplets and place the insert sideways onto a piece of filter paper.
Let the inserts air dry. Prepare the membrane for imaging by labeling a clean glass microscope slide for each insert, and placing a small drop of microscope immersion oil into the center of each slide. Using a scalpel, cut around the perimeter of the membrane on the inside of the plastic insert to detach the membrane.
Use forceps to remove the membrane and place it on top of the oil drop on the labeled slide. Using a compound microscope, view cells at five times, 10 times, or 20 times magnification. For quantification, take multiple non-overlapping images at 10 times magnification.
Determine the total number of invading cells or cells per area for all samples. For each experiment, perform each condition in the assay with three replicate inserts and repeat multiple times for statistically useful results. In this study, in vitro invasion through a protein matrix is used to assess the aggressive phenotypes in oncogenics cell behaviors of mouse mammary tumor cells with altered expression of the zinc finger protein, Zc3h8.
Higher levels of Zc3h8 expression is seen in tumor cell lines or by promoter mediated expression from a plasmid, resulting in rapid rates of cell proliferation, fast migration, growth in 3D environments, and increased encroachment within the in vitro invasion assay. Conversely, decreased expression by shRNA constructs results in less aggressive proliferation, migration and invasion. While the cell invasion decreases upon shRNA knockdown of Zc3h8 expression, that invasion is rescued when the expression is rescued.
The in vitro invasion assay technique is extremely useful as it is a rapid, inexpensive, flexible, and relatively easy approach to studying cell behavior. Cells grown in culture are easy to manipulate genetically. They can even be tested for specific mutations.
The in vitro invasion system also allows for the analysis of small molecule inhibitors as well biological agents like micro RNAs for their effects on cell migration and invasion. This assay also includes a great amount of flexibility, allowing for the alteration of the protein content of the matrix, pore size and chemoattractant.
