Using the Dot Assay to Analyze Migration of Cell Sheets

Podsumowanie

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Streszczenie

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Wprowadzenie

Migration assays are widely used to evaluate the invasive and metastatic potential of tumor cells in vitro. Most commonly, the wound or scratch assay is used to assess migration of epithelial sheets into a cell cleared area^1,2,3. To perform the scratch assay, cells are plated into a monolayer and a "scratch" or cell-free area is created with a pipette tip. The scratch assay is easy to set up with commonly available tissue culture supplies and can be performed in multi-well plates, allowing for processing of multiple samples. However, as the scratch is made, cells are physically removed from the monolayer and often undergo cell death. Furthermore, extracellular matrix attached to the plate is often damaged during the scratching process. Similarly, the use of silicon inserts (such as Ibidi chambers⁴) or of stencils^5,6,7 can lead to mechanical disruption of the cells and the partial removal of matrix proteins used for coating the plates. Another disadvantage of assays monitoring closure of wounds or scratches is their limited time course, as cell migration can only be analyzed until the scratch is closed.

In performing the dot assay, cells are plated as a circular colony onto a coated or uncoated plate⁸. The rationale for this plating strategy is to obtain cell sheets with defined edges, that can migrate or invade into the surrounding cell-free areas without disturbing the culture by removal of cells or inserts. The overall goal of the dot assay is to observe migration of cell sheets as measured by edge displacement or colony diameter, as well as to perform time-lapse imaging to analyze the migratory phenotype of cells in higher spatio-temporal resolution.

Cell migration can be affected by a variety of microenvironmental cues like chemokines, cytokines, and growth factors such as EGF. EGF is a growth factor that exerts its biological effects via binding to its receptor, EGF receptor⁹, and increases invasive and metastatic behavior of tumor cells^4,9,10. Here, the dot assay is used to study EGF stimulated cell migration in a human invasive, lung colony forming breast cancer cell line (MCF10CA1a)^8,11,12.

Protokół

1. Coating of Dishes (Day 1)

NOTE: Make sure not to leave fingerprints or dirt on the bottom of the plate when handling it.

1. Thaw mouse collagen IV on ice, and dilute it with 50 mM HCl (pH 1.3) to prepare 3 mL of a 10 µg/mL collagen IV solution.
   NOTE: Collagen will precipitate at 37 °C. It is therefore important to keep the collagen solution at a low temperature while thawing and working with it. Avoid repeated freeze-thaw cycles by preparing appropriately sized aliquots and storing them at -80 °C.
2. Add 250 µL collagen IV solution to each well 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. Incubate the plates overnight at room temperature.
   Note: As only the growth area needs to be coated, it is sufficient to only cover the cover slip, which is attached to the bottom of the plate, with collagen solution (see Figure 1A, B for a schematic of plate).
3. Next morning, rinse plates with deionized H₂O twice to remove non-absorbed collagen and buffer. Direct the water to the edge of the plate well, not to the edge formed by the plate bottom and the coverslip (if water is pipetted into this area it will splash). Air-dry the plates in the laminar flow hood. Use plates immediately or store at 4 °C for up to 5 days.
   Note: Different cell lines might require different coating, such as fibronectin or collagen I.

2. Plating the Dot Assay (Day 2)

1. Preparing the cell suspension
   1. To prepare cell suspension, use cells that have been grown in a 60-mm tissue culture dish in DMEM/F12 medium supplemented with 5% horse serum to 80% confluence
   2. Rinse the cells with calcium- and magnesium-free Dulbecco's phosphate buffered saline (DPBS) once, add 500 µL trypsin-EDTA (room temperature), and incubate the cells for 3 - 5 min in an incubator at 37 °C, 5% CO₂, humidified atmosphere.
   3. Suspend the detached cells in 4.5 mL culture medium to stop trypsin activity and count a 20 µL aliquot of the cell suspension in a hemocytometer.
   4. Pellet the remaining cells in a 15 mL conical tube by centrifugation at 200 x g for 3 min, aspirate the supernatant, and resuspend the cells in culture medium to 3 x 10⁶ cells/mL.
      NOTE: For other coating substrates and/or cell lines, the optimal cell density must be established in a dilution series. 3 x 10⁶ cell/mL is recommended as a starting point.
2. Plating cells
   1. Place a 10 µL drop of cell suspension at the center of each cover slip of the collagen IV coated 12-well glass bottom plate without touching or scratching the coating (Figure 1A, B). Incubate the plate for 30 min at 37 °C, humidified atmosphere, to allow the cells to attach. Check in an inverted microscope that the cells are attached.
      NOTE: This can be best seen at the edge of the drop, where formation of a monolayer can be observed (Figure 1C). If necessary, incubate the plate up to 3 h. Be sure that the humidity in the incubator is high enough as the drops can dry out quickly. If a reduction of the drop volume during this step is observed, add PBS to the spaces between the wells to increase humidity.
   2. Gently wash the wells with 1 mL of culture medium twice to remove non-attached cells. Add 1 mL culture medium to each well. Check in an inverted microscope that no floating cells remain, otherwise wash the wells again. Incubate cells overnight at 37 °C, 5% CO₂, humidified atmosphere, to obtain well-defined cell sheets.
      Note: Floating cells are likely to re-attach to the plate and to form undesired cell colonies.

3. Stimulating Cells (Day 3)

1. Check all wells using an inverse microscope to ensure that cell colonies have grown properly. If necessary, mark unsuitable wells and do not use them.
2. Label each well of the plate appropriately for each condition investigated. Run each condition in duplicate or triplicate.
3. Change the medium to starve the cells in DMEM/F12 containing 0.1% horse serum (starving medium) 3 h before cells are stimulated.
4. Stimulate the cells by addition of EGF (5 ng/mL final concentration) or other desired mediators and gently mix the medium using a 1 mL micropipettor or by swirling the plate.
5. Incubate the plate for 1 - 4 days to observe edge displacement and edge contour as described in section 4.1 or perform time-lapse imaging as described in section 4.2.

4. Visualization of Cell Colonies and Cell Migration

1. Hematoxylin-eosin (HE) staining to measure colony growth (Day 4 - 7)
   1. Fix cells in 70% ethanol (1 mL/well) for ~2 min.
   2. Stain nuclei with hematoxylin (1 mL/well), ~2 min.
   3. Wash cells with tap water (1 mL/well) until nuclei are blue, 5 - 10 min.
   4. Stain cytoplasm with eosin (1 mL/well), ~2 min.
   5. Rinse with deionized water (1 mL/well).
      Note: Hematoxylin and eosin solutions can be collected and used multiple times until the staining becomes faint.
   6. Air-dry plate.
   7. Flip the plate and measure the dot diameter with a ruler. Alternatively, take pictures of the plate with a camera, and analyze the dot diameter in ImageJ.
2. Time-lapse microscopy (Day 3)
   1. Switch on the incubator microscope and adjust the temperature to 37 °C. Fill the humidifier with dH₂O. Adjust the CO₂ to 5%. Ensure that the incubator chamber is humidified and that the motorized stage can move freely. Close the incubator chamber and allow the microscope to adjust to 37 °C.
      NOTE: The microscope (see the Table of Materials) used here should be heated to 37 °C for at least 3 h before the cells are imaged to avoid drifting of the focus.
   2. Place the plate on the stage of the incubator microscope and set up the stage list. Image two opposing edges and the center of each cell dot (Figure 1A).
   3. Take images every 3 min, for 15 - 24 h using the 10X objective. Download the data and proceed with image analysis in a program of choice, e.g., ImageJ or Matlab.

Wyniki

The dot assay presented here (Figure 1) was performed using invasive, lung colony forming breast cancer cells (MCF10CA1a) as a model system. The dot assay yields highly reproducible cell dots even in the hand of beginners, making it a convenient and easy to execute assay (Figure 1D). The dot assay combined with HE staining, or time-lapse imaging and subsequent particle image velocimetry (PIV) allows the stud...

Dyskusje

During tumor progression cells migrate away from the tissue of origin to invade the surrounding tissue and metastasize to distant sites¹⁵. Migration of cells away from a cell colony can be observed in the dot assay. Here, the dot assay is illustrated by analyzing the migratory phenotype of human breast cancer cells in response to EGF.

To obtain accurate and replicable results several steps in the set-up of the dot assays are critical. First, the coating of the plate det...

Materiały

Name	Company	Catalog Number	Comments
MCF10CA1a cells	Karmanos Research Institute	N/A	cells used for assay
mouse collagen IV	BD Biosciences	354233	used to coat plates
trypsin / EDTA	Thermo Fisher	25300054	used to remove cells from tissue culture plates
DPBS	Mediatech	21-031-CV	used to wash cells
DMEM / F12	Thermo Fisher	11320082	used as culture medium
horse serum	Thermo Fisher	26050088	used to supplement culture medium
12 well glass bottom plate	MatTek	P12G-1.5-14-F	used to grow cells for imaging
EGF (epidermal growth factor, human recombinant)	Peprotech	AF-100-15	used to stimulate cells
fatty acid free BSA	Sigma	A8806-1G	used to make buffer for EGF
hematoxylin	Sigma	HHS32	used to stain colonies
eosin	Electron Microscopy Sciences	26051-10	used to stain colonies
37 °C, 5% CO2 incubator	Sanyo	model MCO-18AIC (UV)
Zeiss AxioObserver with incubator chamber and motorized stage	Zeiss	model Axio Observer.Z1	used for time lapse imaging
ORCA Flash LT sCMOS camera	BioVision	C11440-42U-KIT	used for timelapse imaging in combination with Zeiss AxioObserver
Metamorph	BioVision	MMACQMIC	software used for time lapse imaging
inverted microscope	Olympus	model IX70	used to count cells and to check colonies in tissue culture
plastic box	Lock&Lock	N/A	used as humid chamber (any tight closing plastic box that fits the plates can be used)