Using the Dot Assay to Analyze Migration of Cell Sheets

Summary

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.

Abstract

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.

Introduction

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, c....

Protocol

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 size....

Results

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 study of .......

Discussion

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.......

Materials

List of materials used in this article

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)