The overall goal of this experiment is to report and compare the two assays used to study cell invasion and migration, the Boyden chamber assay, and an optimized in vitro video microscope-based scratch wound assay. These experiments can help answer key questions in the study field of invasion and migration such as the choice of the most relevant method. The main advantage of this optimized in vitro video microscopy-based scratch wound assay is that it provides a reproducible and precise comparison between treatment conditions.
Though this method can assess cell migration and invasion, it can also be applied to other study fields such as healing or tissue regeneration. Prepare SQ20B cells 72 hours prior to day one of the assay by seeding two times 10 to the 1/6 cells in 25 milliliters of culture medium in a 175-square centimeter flask. Allow the cells to grow to 80%cell confluence at 37 degrees Celsius.
On day one after trypsinization and cell counting, seed the cells in a 25-square centimeter flask at a cell density of six times 10 to the 1/5 cells in three milliliters of culture medium for each condition to be evaluated. Incubate the cells at 37 degrees Celsius for 24 hours. On the following day, starve the cells by replacing the medium in each flask with three milliliters of low fetal bovine serum medium.
Starve the cells in the 37 degrees Celsius incubator for 24 hours. For the invasion assay prepare the coated Boyden chambers 12 hours after beginning cell starvation. Place each Boyden chamber in the companion plate.
Add 500 microliters of starvation cell medium to each coated chamber. On the following day, day three, use the companion plate to prepare the chemoattractant. Fill each well of the 24-well companion plate with 750 microliters of complete medium with 10%FBS, hydrocortisone, penicillin and streptomycin.
Transfer the upper chamber into the prefilled companion plate taking care to avoid bubbles. For the invasion assay, carefully remove 450 microliters of culture medium from each Boyden chamber. After trypsinization and counting of the starved SQ20B cells, seed three times 10 to the 1/4 cells in 500 microliters of 0.1%BSA medium giving a final dilution of six times 10 to the 1/4 cells per milliliter.
Place the Boyden chamber in the 37 degrees Celsius incubator for 24 hours. On day four remove each insert from the companion plate and carefully remove the cells from the upper chamber using a cotton swab. Subsequently fix and stain each insert individually to obtain May-Grunwald Giemsa coloration using a staining kit per the manufacturer's instructions.
Perform microscopic analysis on day five. Insert the companion plate with the inserts onto a 20X phase contrast microscope and count each migrated cell on the lower part of the membrane. 72 hours before day one of the assay seed two times 10 to the 1/6 SQ20B cells in 25 milliliters of culture medium in a 175-square centimeter flask and allow the cells to grow to 80%cell confluence.
On day one after trypsinization and cell counting generate a pre-diluted sample of the SQ20B cells at a density of four times 10 to the 1/5 cells per milliliter. Dispense 100 microliters of the cell solution into each well of a 96-well plate to give a final density of four times 10 to the 1/4 cells per 100 microliters in each well. Place the plate in the 37 degrees Celsius incubator and allow the cells to adhere to the plate for 12 to 16 hours.
On the following day take the plate to the hood for wounding using a commercial wounding device. Remove the top of the wound maker and set it in the wash boat solution. Insert the plate into the base plate holder and remove the plate cover.
Replace the pin block by guiding the rear dowels of the pin block into the rear holes of the base plate. Push and hold the black lever and lift the pin block while continuing to hold the black lever down. Using a pipette with an adapted conical tip immediately remove the medium from each well taking care not to touch the wound.
Wash the cells by adding to each well 100 microliters of cell medium warmed to 37 degrees Celsius. After the second wash, use a pipette with an adapted conical tip to aspirate the cell medium. Next, add 100 microliters of medium specific for each treatment condition to each well making sure to avoid creating any bubbles in the wells.
Place the 96-well plate into the adapted rack of the video microscope. Using the video microscope software program the schedule of scans at one image per well. For a migration invasion experiment a maximum two-hour interval is required.
If the objective of the experiment is to produce a video a maximum 30-minute interval is preferred. Monitor and check the cell migration for a minimum of 24 hours and up to five days using an appropriate cell mask adapted for each cell type to analyze cell migration. To obtain a cell mask adapted for each cell line, generate a cell processing definition from the software using a specific cell image collection.
Trace the curves and export the data to a spreadsheet which can be used to analyze and compare the results. To prepare for the cell invasion assay seed SQ20B cells in the same way as demonstrated for the cell migration assay, and incubate the 96-well plate in the incubator. On day two of the assay prepare the extracellular matrix.
Defrost the matrix at four degrees Celsius for at least 12 hours before use and make sure no aggregates are visible. Chill the microcentrifuge tubes on ice for five minutes. Dilute the matrix with pre-cooled to four degrees Celsius conical tips in the pre-cooled microcentrifuge tubes with cell medium cooled to four degrees Celsius to obtain a final concentration of 300 micrograms per milliliter.
Keep the microcentrifuge tubes with pre-diluted matrix in the refrigerator at four degrees Celsius. Retrieve the 96-well plate from the incubator. Under the hood make the wound using the commercial wounding device according to the manufacturer's protocol as demonstrated earlier.
Use a pipette with an adapted conical tip to immediately remove the medium from each well and be careful not to touch the wound. Wash the cells twice using 100 microliters of cell medium warmed to 37 degrees Celsius. After the second wash place the plate onto ice for five minutes to equilibrate its temperature.
Remove the cold medium from each well taking care to pipette carefully from the edge and to avoid touching the wound. Using conical tips pre-cooled at four degrees Celsius add 50 microliters of pre-diluted matrix to each well. Place the plate in the 37 degrees Celsius incubator for 30 minutes.
After 30 minutes add 100 microliters of the adapted cell medium to each well as indicated for each treatment condition. Place the plate into the adapted rack in the video microscope. Subsequently program the scans and perform video microscopy analysis as demonstrated for the cell migration assay.
Representative results of a Boyden membrane after cell-fixation are shown. The sub-optimal membrane has cell clusters on the upper side of the membrane and uninterpretable results. In contrast, the optimal membrane has countable cells in the lower part of the membrane stained in blue.
An optimal result of the scratch wound assay is illustrated by these images of wound healing at zero hour, 15 hours and 30 hours. The criteria for a quality experiment are a linear wound, no cell fragments observed in the wound, and optimal cell confluence. 90%confluence is the optimal cell density for the wound healing assay as shown in panel A.Panel C shows an example of low cell density and panel B shows cell clusters caused by insufficient washing of the cell pellet.
This graphical representation of wound healing obtained using the video microscope software shows quantification of parameters of wound cell confluence according to time for four treatment conditions. It was observed that the combination treatment significantly decreases cell migration. After watching this video you should have a good understanding of how to evaluate cell migration and invasion process.
