The overall goal of the dot assay is to assess the migratory phenotype of attached cohesive cell sheets in response to microenvironmental cues. This method can help answer key questions in the cancer field, such as how cytokines or growth factors alter tumor cell motility. The main advantage of the dot assay is that it is easily performed and used for reproducible results.

Though this method can provide insight into cell motility, it also lends itself to a range of subsequent assays, such as staining of cells to assess growth colony morphology and cell scattering, immunostaining, protein and DNA or RNA analysis, or injection of cells into animals. To begin, thaw mouse collagen four on ice, and dilute it with 50 micromolars HCL to prepare three milliliters of a 10 microgram per milliliter collagen four solution. Add 250 microliters of collagen four solution to the wells of 12-well glass-bottom plates, and place them into an appropriately sized, tight-closing plastic box.

Place wet paper towel over and around the plates to manufacture a humid chamber, and close the box. Then, incubate the plates overnight at room temperature. The next morning, to move non-absorbed collagen and buffer, use deionized water to rinse the plates twice, directing the water to the edge of the plate well, rather than the edge formed by the edge bottom and the cover slip.

Air dry the plates in a laminar flow hood, and use them immediately or store them at four degrees Celsius for up to five days. To prepare cell suspension, use cells that have been grown in a 60 millimeter tissue culture dish in DMEMF12 medium, supplemented with 5%horse serum to 80%confluence. Use calcium-and magnesium-free DPBS to rinse the cells once, then add 500 microliters of room temperature trypsin EDTA, and incubate the cells at 37 degrees Celsius in 5%CO2 in a humidified atmosphere for three to five minutes.

Suspend the detached cells in 4.5 milliliters of culture medium to stop trypsin activity, and use a hemocytometer to count a 20 microliter aliquot of the cell suspension. Pull out the remaining cells in a 50 milliliter conical tube by centrifugation at 200 times gravity for three minutes. Then, aspirate the supernatant, and re-suspend the cells in culture medium to three times 10 to the six cells per milliliter.

To plate the cells at the center of each coverslip of the collagen four coated 12-well glass-bottom plate, place a 10 microliter drop of cell suspension, taking care not to touch or scratch the coating. Plating of a non-circular drop will not yield a circular cell sheet, and if the coating is touched or scratched, it will prevent the cells from adhering well. Incubate the plate at 37 degrees Celsius in a humidified atmosphere for 30 minutes, to allow the cells to attach.

Then, check for attachment under an inverted microscope. With one milliliter of culture medium, gently wash the wells twice, to remove non-attached cells, then add one milliliter of culture medium to each well. Check under an inverted microscope that no floating cells remain, otherwise wash the wells again.

Incubate the cells overnight at 37 degrees Celsius in 5%CO2 in a humidified atmosphere to obtain well-defined cell sheets. Using an inverted microscope, check all the wells to ensure that cell colonies have grown properly. If necessary, mark unsuitable wells and do not use them.

Label each well of the plate appropriately for each condition investigated. Run each condition in duplicate or triplicate. Change the medium to starve the cells in DMEMF12 containing 0.1%horse serum, three hours before cells are stimulated.

Then, stimulate the cells by adding EGF or other desired mediators. Use a one microliter pipetter, or swirl the plate gently to mix the medium. Incubate the plate for one to four days to observe edge displacement and edge contour.

To carry out the H and E staining, wash the cells with PBS twice and fix them for approximately two minutes. Add hematoxylin for approximately two minutes to stain the nuclei. Wash the cells with tap water for five to 10 minutes, until the nuclei are blue.

Then, stain the cytoplasm with eosin for two minutes before rinsing the cells with deionized water. Air dry the plate, then flip it over and use a ruler to measure the dot diameter. Alternatively, take pictures of the plate with a camera, and use ImageJ to analyze the dot diameter.

To carry out time lapse microscopy, switch on the incubator microscope, and adjust the temperature to 37 degrees Celsius. Fill the humidifier with DH2O, then adjust the CO2 to 5%Ensure that the incubator chamber is humidified, and that the motorized stage can move freely. Then, close the incubator chamber and allow the microscope to adjust to 37 degrees Celsius.

Place the plate on the stage of the incubator microscope and set up the stage list, then image two opposing edges and the center of each cell dot. Using a 10X objective, take images every three minutes for 15 to 24 hours. Download the data and proceed with image analysis in a program of choice such as Matlab.

Initially, phase-contrast imaging of cell dots revealed that invasive, lung colony-forming breast cancer cells maintain a mesenchymal phenotype after stimulation with EGF. However, single cells leaving the sheet are observed more frequently in EGF-stimulated cell dots. The response of lung colony-forming breast cancer cells to EGF was further assessed by H and E staining of cultures that were stimulated with EGF for four days.

Indeed, EGF stimulation resulted in an increased colony diameter, indicating that migration of EGF-stimulated cells might be altered. In this dot assay, cells at the sheet edges were imaged for nine hours following stimulation of colony-forming breast cancer cells with EGF. PIV revealed that EGF increased cell speed to 0.63 micrometers per minute from 0.45 micrometers per minute in control cells.

In addition, EGF reduced the variability of cell directionality, or angular spread, from 0.95 in control cultures to 0.79 in EGF-stimulated cultures. In addition, as was observed by H and E staining after four days, EGF increased the radial displacement of the epithelial edge over nine hours, from 257 micrometers to 356 micrometers. Once mastered, the hands-on time for this technique is a couple of hours.

Following this procedure, other methods like time lapse imaging, immunostaining, and DNA and RNA analysis can be performed in order to answer additional questions. For example, it can be used to analyze if proteins are differentially expressed in fast or slow migrating cells, or when cells migrate into the surrounding environment. After watching this video, you should have a good understanding of how to set up the dot assay, and use it to assess the migratory phenotype of attached cohesive cell sheets in response to microenvironemntal cues.