Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of cell movement and invasive capacity of tumor cells is of great interest. This integrated method for investigating cancer cell migration and invasion, on a single platform in real time, provides an easily reproducible and time-efficient option for studying cell mobility and pathology.
This method can provide insight into cancer cell invasion, through the extra-cellular matrix, and can be applied to other cell types. To achieve an optimal scratch time, ensure that the seeding density has been optimized and do not prolong the incubation period once the cell monolayer has reached 100 percent confluence. To determine the optimal number of seeded cells for a migration assay, place the cells in a range of densities in triplicate in a 96-wall plate.
Place the plate into the live cell imager, and select schedule scans from the task list in the imager software. In the drawer setup pane, determine the positions of the plate, and click add vessel'to select the plate type. In the scan setup pane, select or edit the scan pattern according to the experimental plate setup, and set the scan type as standard.
Right-click on the timeline, and select set intervals. Then set add scans every'to two hours for 24 hours, and click apply. After 24 hours, open the drawer setup to allow the experimental plate to be selected, and click remove vessel.
Select three to six representative images, and place them in a new image collection. To determine a proper processing definition, use segmentation adjustment, clean-up, and filters to apply an appropriate confluence mask, and use preview current all'to view the accuracy of the masks. Launch the analysis job and determine an optimized cell density, according to an approximate 100 percent confluence against time, within six to eighteen hours, depending on when the migration assay commences.
Then, apply the confluence processing analysis tool to the high-definition phase contrast images automatically collected to generate a cell proliferation curve against time. Before beginning the assay, coat the plate for the invasion assay with 50 microliters of extracellular matrix gel, diluted in ice-cold cell culture medium, to a 100 microgram per milliliter concentration. Then, place the plate in a cell culture incubator overnight.
The next afternoon, gently aspirate the excess medium, and plate the cells at the optimized density, in triplicate, into the appropriate wells of the extracellular matrix-coated plate, and into an additional uncoated 96-well plate. Then, place the plates in the cell culture incubator overnight. The next morning, place the uncoated migration assay plate into the base plate holder of the scratch tool, and use the guiding dowels to carefully place the top of the holder on to the base.
Press and hold the black lever while carefully lifting the pin block to make the scratches. The scratches in each well should be visible with the naked eye, as well as under the microscope. Then, wash the plate one or two times with pre-warmed culture medium to remove any detached cells or cell sheets.
For the invasion assay, scratch the coated plate as just demonstrated. After removing any detached cells and sheets, use a pre-chilled cool box to equilibrate the plate at four degrees Celsius for five minutes before carefully aspirating the cold medium. Next, add 50 microliters of diluted extracellular matrix gel into the appropriate wells and place the plate into the cell culture incubator for thirty minutes.
Bubbles are common when working with extracellular matrix gel, but they can be eliminated by aspirating 70 percent ethanol. Then add 100 microliters of fresh, warm medium, with or without the test compound, to the appropriate wells of either the migration or invasion assay plate, and place the plate into the live cell imaging platform. To clean the scratch tool, place the top pin block in individual 45 milliliter wash boats, containing 5 percent detergent one, one percent detergent two, sterile distilled water, or 70 percent ethanol for five minutes per wash before placing the scratch tool back on to its base plate, for storage in a dust-free environment.
For imaging the wound assays, after allowing the plate to equilibrate for five minutes, select vessel type'as the image log plane, and set the scan type as scratch wound. Select or edit the scan pattern, according to the experimental plate setup, and schedule 24 hours of repeat scanning every one to two hours for 72 hours, or until the wounds are healed as demonstrated. When all of the wounds have healed, stop the scan, and select three to six representative images, spanning a range of sound percentages, including images right after the wound has been made, and wound closure by 10 and 50 percent.
To determine a proper processing definition, apply an appropriate scratch wound mask, and a confluence mask as demonstrated, and use preview current all'to view the accuracy of the masks. Then, launch the analysis job. In a migration assay, the wound width is the average distance between the cell sheets beside the wound.
The wound confluence is the confluence within the wounded area. The ideal initial wound confluence should be close to zero percent. The relative wound density and wound confluence suggest the speed of the cells occupying the scratch wound area.
These data almost overlap in both of these representative cell lines. Different cell lines demonstrate very different wound healing abilities, indicating differential migratory abilities between the cell lines. For example, lamellipodia are observed in this breast adenocarcinoma cell line at the migration front at the beginning of the migration, whereas this mucin 1-producing breast cancer cell line exhibited no sign of cell migration at the same time point.
Further, the relative wound density of the breast adenocarcinoma line was saturated after 50 hours, whereas the relative wound density of the mucin 1-producing breast cancer cell line did not change over time, underscoring the highly invasive phenotype of the adenocarcinoma cells and and non-invasiveness of the mucin 1-producing breast cancer cells. The adenocarcinoma cells also behaved differently while invading through the extracellular matrix gel, demonstrating an elongated morphology with leading protrusions, and in migration assays, a bleb at the migration front was visualized. When plating, remember that if too few cells are seeded, the scratch will not form properly, and if too many cells are seeded, there will be overcrowding.
Following this procedure, track treatments can be added, allowing this method to be adapted for high-throughput track screening. This method allows researchers to perform these assays on a single platform, which is high-throughput, and allows researchers to monitor their cells in real time, rather than in experimental endpoint. The disinfectants used for sterilizing the scratch tool may be hazardous.
Make sure you collect and dispose of the solutions according to the relevant MSDS, and the regular trigger lines of your facility.
