In vitro Cell Migration and Invasion Assays

Summary

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Abstract

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

Introduction

Motility is an essential feature of live cells. Cell migration is involved in the conception of life, embryonic development, immune response, and many pathological processes such as cancer metastasis and inflammation^1-9. Therefore, methods to study cell migratory behavior are very useful research tools for a wide range of disciplines in biomedical sciences, biology, bioengineering, and related fields.

The study of cell migration in cancer research is of particular interest as the main cause of death in cancer patients is related to metastatic progression. In order for cancer to spread and disseminate throughout the body, cancer c....

Protocol

1. Cell Culture Wound Closure Assay

1. Detach cells from the tissue culture plate using 0.25% Trypsin-EDTA solution. Pellet cells in a 15 ml conical tube by centrifugation, aspirate the supernatant, and re-suspend cells in culture media. Plate the appropriate number of cells in a 6-well plate for 100% confluence in 24 hours.
   Note: Tests may be needed to determine the time and number of cells to achieve 100% confluence due to the cell type and the size of the well being used (for example, 1 x 10⁶ B16F10 melanoma cells are seeded in a well of a 6-well plate 24 hours prior to the wound generation). Alternatively, 12-well or 24-well plates ....

Results

The wound closure assay and the transwell cell migration assay presented here were performed using mouse B16F10 melanoma cells as a model system. In the wound closure assay, B16F10 cells were seeded in a 6-well tissue culture plate and grown to 100% confluence in 24 hours. A wound of approximately 700 µm wide was generated using a pipette tip and the wound closure (cell migration) was recorded using the time-lapse microscopy. Alternatively, wound closure can also be studied by taking snapshot pictures at different time p.......

Discussion

Cell migration is an important aspect to study in cancer research and it can also be applied to developmental, immunological and wound healing studies. The cell culture wound closure assay and the transwell cell migration and invasion assays reveal detailed information of cell migratory behaviors and can be used to investigate the molecular mechanisms of cell migration^1,2,10,14. Our study used these cell motility assays to determine the migration velocity and invasion capabilities of a B16F10 melanoma cell lin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco’s modified Eagle medium (DMEM)	Gibco	11995-073
Fetal bovine serum (FBS)	Gemini	100106
Bovine serum albumin (BSA)	Sigma-Aldrich	A4503-50G
Trypsin EDTA 0.25%	Gibco	25200-056
Dulbecco’s phosphate buffered saline (DPBS)	Gibco	14190-250
Crystal violet	Sigma	C0775-100G	Dissolved in water at 0.2%
Cell disassociation buffer	Gibco	13151-014
Cell culture incubator	Thermo Fisher Scientific		Model # 3145
SterilGARD biosafety hood	The Baker Company, Inc.		Model # VBM-600
EVOS Fl inverted microscope	Thermo Fisher Scientific		Model # AMF-4302-US
Tissue culture plate	Becton Dickinson	353046	Catalog number varies depending on the type of culture plate
Corning Transwell insert	Fisher	07-200-150	Catalog number varies depending on the pore size of the membrane