In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

1. Preparation of a Biosafety II Cabinet (BSC)

NOTE: The methods described in this protocol should be conducted with personal protective equipment, while using good laboratory practices to achieve aseptic technique.

1. Lift the BSC protective glass to operating height and turn on laminar flow fans.
   1. Use 70% isopropanol aqueous solution to perform a sanitation wipe of the working area. Wipe BSC from the back wall and side walls and work down to the base plate.
   2. Wipe all materials that will be used in the assay with 70% alcohol (isopropanol or EtOH) and place them inside the BSC.

2. Human Dermal Fibroblasts Seeding in a T75 Flask

1. Obtain vials with the desired cryopreserved cell line (neonatal human dermal fibroblasts [hDFn], at cell concentration of 500,000 cells/vial, and at passage p3–p5).
   1. Place a vial with cells in a 1.0-cm-deep water bath at 37 °C to allow thawing. Thaw vial until approximately 70% of cell solution is liquid. Wipe down vial with 70% alcohol and place vial in vial rack inside BSC.
      NOTE: Ensure water does not come in contact with threads or cap of vial to prevent potential contamination during cell seeding. Prevent complete thawing of cells in water bath as failure to do so may cause unintentional cell death.
2. Draw out 10 mL of media supplemented with 10% Fetal Bovine Serum (FBS). Use an automatic pipettor and 10 mL pipette tip to aspirate into T75 tissue culture flask. Allow media to fully saturate the base of the flask by gently rocking the flask back and forth.
   1. Open vial with cell stock and aspirate solution from the vial, being mindful of pipetting speed, as cell membranes will be vulnerable from cryopreservation.
   2. Transfer cells to a T75 tissue flask with media. Eject thawed cell into tissue flask, again being mindful of pipetting speed.
      NOTE: Seeding density of cells in tissue flask is approximated to be 6,600 cells/cm². Seeding density can be altered to accommodate the user and desired timing of experimentation.
   3. Measure out 1 mL of media from T75 tissue flask and transfer volume back into the vial. Transfer volume from the vial back into the T75 flask.
      NOTE: Performing this step helps maximize cell yield from the vial.
   4. Tighten the tissue culture flask cap and gently rock to uniformly disperse cells in suspension across the entire surface. Check for even distribution of cells using an inverted microscope.
3. Label flask with cell type, date, initials, lineage, and purpose (e.g., hDFn, 07/11/2018, NDC, Passage 4, Arsenic Scratch Assay). Place tissue culture flask in a humidified incubator at 37 °C and 5.0% CO₂ for 72 h.
   NOTE: Growth medium should be changed 12-24 h after initial seeding to remove trace amounts of dimethyl sulfoxide (DMSO) cryopreservative, and then changed every 48 h after that to ensure cells are properly nourished. The incubation and growth period will depend on the calculated number of cells needed for the scratch assay. The doubling time for hDFn cells is typically 16-24 h under normal conditions. However, doubling rate may change due to elevation, pH, temperature, humidity, medium type, etc., and must be accounted for when planning experiments.

3. Cell Counting and Subculture into 12-Well Tissue Culture Plate

1. Retrieve the T75 tissue culture flask from incubator. Observe adhered cells using an inverted microscope at 10X objective magnification (100X total magnification) and determine the approximate cell confluence.
   1. Scan the majority of the tissue culture flask when observing confluence to ensure the most representative percentage is approximated. Evaluate individual cell morphology for any abnormalities, or for bacterial and/or fungal contamination.
2. Assemble the automatic pipettor with a 10 mL pipette tip. Use pipettor to aspirate the full volume of media from the T75 and transfer solution to a waste container. Prevent the pipet tip from contacting the cell adhering surface. Discard pipette tip in a biohazard bin.
   1. Assemble the automatic pipettor with a 5 mL pipette tip. Aspirate 5 mL of the balanced salt solution, dispense in the flask, and gently rock the flask to rinse excess media, dead cells, and waste product.
   2. Draw the volume from the T75 and dispense into the waste container. Discard the pipette tip into the biohazard bin.
   3. Assemble the automatic pipettor with a 5 mL pipette adapter. Aspirate 2 mL of trypsin from stock, dispense in the flask, and gently rock the flask to disperse the enzyme over the adhered cells. Ensure complete saturation of the adherent surface. Place the tissue flask in the incubator for 2-3 minutes (min).
      NOTE: The maximum enzyme-substrate reaction should not exceed 10 min.
   4. Observe the tissue flask under an inverted microscope at a 10X objective lens magnification (100X total magnification). Apply gentle vibrations to facilitate cell detachment.
   5. Wipe the T75 flask with 70% alcohol and place inside the BSC.
   6. Assemble the automatic pipettor with a 5 mL pipette tip. Aspirate 5 mL of media from the stock and add to the T75 tissue flask to stop the enzyme-substrate reaction. The media:trypsin ratio should be 3:1. Pipet up and down the base of the flask 2-3 times to rinse and ensure that the reaction has stopped. Aspirate the complete volume of fluid from the flask and transfer into a sterile 15 mL conical tube.
   7. Fill a second 15 mL conical tube with an identical volume of media.
   8. Place both 15 mL conical tubes in an antipodal position to one another. Apply 200 g of centrifugal force for 5 min at 25° C by setting equipment’s running parameters.
   9. Wipe the 15 mL conical tube with pelleted cells with 70% alcohol and place inside the BSC.
   10. Use the automatic pipettor with a 5 mL attachment to pipet off media, leaving behind approximately 250 µL of fluid and cell pellet. Pay attention to the location of the pipet tip, as the cell pellet should remain undisturbed. Dispense the volume collected into a waste container and discard the tip in a biohazard bin.
   11. Use a microcentrifuge tube rack to run the bottom of the conical tube across the vial slots, creating a swift vibration, to disturb and re-suspend the cell pellet (sometimes called “rack raking”).
       NOTE: Avoid using a vortex mixer to perform this step. Gravity forces created by vortex mixers exceed cell g-force thresholds and can cause lysing. When completed correctly, the new cell solution will appear as a cloudy mixture with no remaining pellet. Diligence performing this step will oftentimes create a single cell suspension, leading to increased accuracy when counting cells.
   12. Add 2 mL of media to the cell stock or resuspension. Pipet the cells gently down the side of the tube 20 times to break up any clumps. Record the total cell suspension volume and set aside briefly.
3. Pipet 20 µL of Trypan Blue stock (0.4%) into an empty well of a 96-well plate using a sterile pipet.
   1. Mix the stock cell solution to generate a uniform cell suspension before transfer. Use a sterile pipet tip to remove 20 µL of cell stock (avoiding touching the tube walls). Add to the 20 µL of Trypan Blue solution in the 96-well plate.
   2. Pipet gently to mix contents. Pipet 10 µL of the mixture between the coverslip and the counting chamber on the hemocytometer.
   3. Observe the cells and the grid under a 10X objective. Observe nine large squares within the field of view. Count cells only in the center and 4 corner squares (total of 5 squares).
      NOTE: Look for a uniform distribution of cells across the entire grid to ensure an accurate count.
   4. Tally all cells (transparent and blue) in the 5 identified squares of the grid. Separately, tally only the nonviable (blue) cells in the same 5 squares.
   5. Calculate viable cell count using: 
      where x = viable cell count, a = total cells, b = non-viable cells
   6. Calculate the viable cell density using: 
      where x = viable cell density and y = total sum of viable cells.
   7. Calculate total viable cells: 
      where x = total viable cells, y = viable cell density, f = cell suspension volume (mL).
   8. Calculate %Cell viability: 
      where x = Percent cell viability, y = viable cells counted on grid, f = total cells counted on grid.
4. Mark a horizontal line across the bottom of the 12-well plate to serve as reference marks during imaging.
   1. Use the viable cell density (calculated in 3.3.7) to calculate the volume of media to dilute cell stock in for seeding a 12-well tissue culture plate.
      NOTE: The optimal cell seeding density for hDFn cells is 5,000 cells/cm².
   2. Dilute cell stock to 19,000 cells/mL using media and seed every well with 1 mL of cell stock. Mix cell stock periodically to ensure even cell seeding across all 12 wells.
   3. Label each plate with the cell type, the lineage, the user initials, and the purpose. Place plates in incubator set to 37 °C and 5.0% CO₂. Allow cells to grow until 100% confluence is achieved for scratch assay.

4. Arsenic (As) Stock Solutions and Calculations

1. Dilute sodium arsenite (NaAsO₂) directly into cell culture media with 10% FBS at working concentrations of 10, 1.0, 0.1, and 0.01 µM As.
2. For this, make an initial 1 mM stock As solution by diluting 3.9 mg of sodium arsenite into 30 mL of media. Serially dilute the 1 mM stock for the remaining As solutions.

Table 1. Arsenic stocks and serial dilutions. This table explains the calculations used to create the As stock solutions and working stock solutions for treatment groups.

5. Scratch Assay Technique and Arsenic Contamination

1. Obtain a 12-well plate with cells from the incubator. View cells at a 10X objective magnification (100X total magnification). Determine cell confluence within each well.
   NOTE: Each individual well must reach 100% confluence prior to scratching. This critical step ensures consistency within the assay and helps standardize assay protocols across experiments. If any wells have not reached 100% confluence, allow cells to incubate for additional time until all wells have reached 100% confluence. Additional growth time in wells that have already reached 100% confluence will not be affected. Confluent cells will typically become growth arrested and remain as a monolayer culture, while allowing sub-confluent wells to reach 100% confluence.
2. Assemble an automatic pipettor with a 10 mL pipette tip. Aspirate the growth media out of all wells. Dispose the volume in liquid waste and the pipette tip in the biohazards bin.
   1. Assemble a p200 pipettor with a 1-200 µL sterile tip. In each well, produce a scratch “mock wound” by gliding the tip across cell surface from a 12-o’clock to 6-o’clock location.
      NOTE: Three factors that will greatly affect consistency are speed, pressure, and tip angle. The scratch should be performed swiftly, with a constant pressure, while being mindful of the tip angle staying perpendicular to the base of the plate. Scratching too slowly will cause crooked lines, excess pressure can permanently score cell adhering surfaces, and a tip angle less than 90° will narrow the scratch width. Any of these common mistakes can result in altered migration rates and patterns.
   2. Clear away excess debris and cell clumps by rinsing with 1 mL of balanced salt solution per well. Rock the plate side-to-side to facilitate washing. Remove the balanced salt solution from wells and dispose in a waste container. Dispose of the 5 mL pipette tip into the biohazards bin.

Figure 1: Schematic representation of the scratch assay. All work is done within an aseptic field to decrease the chance of contamination. Cells are seeded at a desired density in tissue culture flasks and grown in an incubator set to human physiologic conditions (5% CO₂, 37 °C). Upon reaching a desired confluence, cells are aseptically sub-cultured into 12-well tissue culture plates at a desired seeding density. Cells are grown to 100% confluence in each well of the 12-well plate and scratched using a sterile pipet tip. This "scratch" creates an in vitro mock wound (denoted by double headed arrow), in which cells naturally migrate to close the open area. Each well can be uniquely conditioned and cellular migration rates can be observed and quantified in response to different treatment conditions. Please click here to view a larger version of this figure.

3. Use a p1000 pipettor with a 1,000 µL tip to pipet 1,000 µL from arsenic stocks into wells that have been randomly assigned with a treatment group. Use a new pipet tip for each well to prevent cross contamination. Record the time when treatments are added. Place 12-well plates in an incubator set at 37 °C and 5.0% CO₂.

6. Imaging

1. Capture images of each well at 10X objective magnification every 4 h over a 24 h period (0, 4, 8, 12, 16, 20, and 24 h). Reference the line previously drawn on the bottom of the plate to image the same location along the scratch within each well at subsequent time points.
   NOTE: It is imperative that the same locations are photographed at each time point to allow for accurate image analysis and to obtain representative data.

7. Image Analysis

1. Upload images captured on the inverted light microscope to a computer and save as a JPEG image with a detailed file name.
2. Open ImageJ.
3. Upload an image of a stage micrometer with known distances to the software to convert pixels to millimeters (mm) when taking image measurements by dragging and dropping the file into the software window.
   NOTE: The micrometer should have lines demarcating a known 1 mm length and the image must be taken at the same magnification used to capture images of cells. For example, in this study images were taken at 100X total magnification, thus the image of the stage micrometer was taken at 100X total magnification.
   1. Click the Straight, Segmented, or Freehand Lines options.
   2. Draw a line of known distance on the micrometer image by using the tick-marks with a length associated to each tick marks. To draw a straight line: click mouse, hold, drag the cursor to desired location, then let go of mouse.
   3. Select Analyze then select Set Scale.
   4. Set Known Distance to the known length of the line drawn in previous step.
   5. Set Unit of Length 세스 mm.
   6. Check the global box.
   7. Select OK to exit out of the menu.
   8. Close out of the stage micrometer image.
4. Upload one scratch assay image into ImageJ by dragging and dropping image file into the window.
   1. Draw a straight line across the width of the scratch (from left leading edge to right leading edge).
   2. Press the letter M on keyboard to record the measurement of the straight line drawn. A small Results window will immediately appear after typing the letter M. This is where the width measurement will be.
5. Repeat step 7.4 a total of 10 times to obtain 10 measurements along the length of the scratch.
   NOTE: It is important to generate the most representational width values down the entire length of the scratch. Make sure to space out the 10 width measurements equally along the length of the scratch from top to bottom.
6. Create a scratch assay spreadsheet file to copy and paste the measurements.
   1. Copy and paste the 10 width measurements from the Results window into appropriate column in a spreadsheet file (Table 2).
      NOTE: When the measurements are copied from the Results window and pasted into the spreadsheet, there will be extraneous columns of values; these values should be deleted, preserving only the 10 width measurements.

Table 2. Raw width measurements table format. This table shows the organizational structure for the raw measurements obtained.

7. Repeat the above step for the remainder of the scratch assay images.
8. Calculate the average of the 10 measurements at each time point (0-24 h) for every well.
9. Determine percent closure calculations using values from step 7.8.
   1. Create a table at bottom of spreadsheet titled “width measurement averages” (Table 3).
   2. Copy and paste the Average of 10 Measurements row calculated in step 7.8 into the “width measurement averages” table.
   3. Create another table next to the Width Measurement Averages table. Title this table Percent Wound Closure (Table 4).
      NOTE: The value in the 0 h column for each well should be 0 because the scratch is 0% closed at the initial 0 h photo for all wells.
   4. Navigate to the next column over (4 h).
   5. Calculate the percent closure for 4, 8, 12, 16, 20, and 24 h time points: 
      
      where, x = percent closure, i = initial scratch width (0 h width), f = final scratch width (4, 8, 12, 16, 20, or 24 h width).

Table 3. Width measurement averages table format. This table shows the organizational structure for the width measurement averages calculated using the raw width measurements in Table 2.

Table 4. Percent wound closure table format. This table shows the organizational structure for the percent wound closure values calculated using the width measurement averages in Table 3.

10. Create another table next to the Percent Wound Closure table. Title this table AUC (Table 5).
    1. Calculate the 0-4 h area under the curve (AUC) value in the 0-4 h column for each well: 
       
       where x = 0-4 h AUC value, a = % closure value for 0 h, b = % closure value for 4 h, h = the time between the two measures (4 h in this study).
    2. Calculate the 4-8 h area under the curve (AUC) value in the 4-8 h column for each well:
       
       Where x = 4-8 h AUC value, a = % closure value for 4 h, b = % closure value for 8 h, h = the time between the two measures (4 h in this study).
       ​NOTE: One AUC value should be calculated for each 4 h time interval over the 24 h period for each well.
    3. Sum all of the AUC values from 0-24 h (calculated in steps 7.10.1-7.10.2) to get a summed AUC value for each well. Ensure these values are properly saved for statistical analysis.

Table 5. AUC table format. This table shows the organizational structure for the AUC values calculated using the percent wound closure values in Table 4.

8. Statistical Analysis

1. Determine the statistical analysis method that best fits both the experimental design and summed AUC values obtained during the assay.