A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Summary

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Abstract

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

Introduction

Cell migration and invasion are important biological processes that enable normal functions in the human body, such as wound closure, invasion of placenta into the uterus and mammary gland morphogenesis^1,2,3. The human body has precise and strict control of these biological events; however, there are some exceptions. Malignant tumors, for example, are able to escape this safeguard, exhibit abnormal proliferation and invade into neighboring tissue, which is called metastasis. Metastasis is the major cause of cancer-related mortality⁴.

Protocol

NOTE: Two cell lines should be handled separately. The following procedures should be applied to one single cell line if not specified.

1. Optimize Cell Density Prior to Wounding

1. Culture adherent cells in T75 cm² tissue culture flasks to about 80% confluence in phenol-red free Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine and 2 µg/mL insulin at 37 °C with 5% CO₂ (standard incubation conditions for most cancer cell lines, culture formulations are cell line-dependent).
2. Remove the culture media by pipetting of....

Results

This migration/invasion assay is based on the wound healing assay, which evaluates the rate of the cells moving into a cell-free area created by the 96-pin scratch tool. The difference between the migration and invasion assays are that migration assays measure cells moving on the tissue-culture treated plastic surface and invasion measures cells moving across ECM gel.

The scratch tool is designed to make consistent scratch wound.......

Discussion

Migration and invasion are important parameters to assess the mobility of cancer cells. By using the 96-pin scratch tool, it is possible to conduct wound healing assays in 2D and 3D simultaneously. Apart from facilitating automatic scanning, providing a stable cell culture environment with minimum disruption, the scratch assay conducted using the 96-pin scratch tool provides consistent scratch wounds, enabling experiments that are more robust and reproducible. The 96-well plate format gives additional options of either i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5% trypsin-EDTA solution (10x)	ThermoFisher Scientific	30028-02	Dilute to 2x in DPBS
Countess II FL Automated Cell Counter	ThermoFisher Scientific	AMQAX1000	Automated cell counter
Detergent 1 (Alconox)	Sigma-Aldrich	242985	0.5% working concentration
Detergent 2 (Virkon S)	VetProduct DIRECT		1% working concentration
Dulbecco’s Modified Eagle Medium, no phenol-red	ThermoFisher Scientific	21063-045	Supplimented with 10% FBS, 200 mM L-glutamine, 2 μg/ml insulin
ECM Gel (matrigel)	Sigma-Aldrich	E6909	Growth-factor reduced, phenol red free
Essen ImageLock 96-well plate, flat bottom	Essen	4379
EVE Counting slides	BioTools	EVS-50
Fetal bovine serum (FBS)	Bovogen Biologicals	SFBS-F-500ml
IncuCyte 96-well scratch wound cell invasion accessories	Essen	4444	Including CoolBox, 2x CoolSink
IncuCyte Cell migration kit	Essen	4493	Including the 96-well pin block, 2x wash boats and the software
IncuCyte ZOOM	Essen		Live cell analysis system
Insulin solution human	Sigma-Aldrich	19278-5ML
L-glutamine solution (100x)	ThermoFisher Scientific	25030-091
Tissue culture flask, 75 cm2 growth area	Greiner Bio-One	658175