An in Vitro FluoroBlok Tumor Invasion Assay

J Vis Exp. 2009 Jul 20;(29):1475. doi: 10.3791/1475.

Abstract

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro(1,2). It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC(12;)(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at >99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

Publication types

  • Video-Audio Media

MeSH terms

  • Animals
  • Collagen / chemistry*
  • Drug Combinations
  • Extracellular Matrix / metabolism
  • Extracellular Matrix / pathology
  • Fluoresceins / chemistry*
  • Fluorescent Dyes / chemistry*
  • Humans
  • Laminin / chemistry*
  • Membranes, Artificial
  • Neoplasm Invasiveness
  • Neoplasms / metabolism
  • Neoplasms / pathology*
  • Proteoglycans / chemistry*

Substances

  • Drug Combinations
  • Fluoresceins
  • Fluorescent Dyes
  • Laminin
  • Membranes, Artificial
  • Proteoglycans
  • matrigel
  • calcein AM
  • Collagen