Page 60 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Kinetic Transendothelial

Migration Assays

Label-free, live cell imaging assay to visualize and
quantify transendothelial migration

Introduction IncuCyte Transendothelial Migration Assay at
®
a Glance

The recruitment of leukocytes to sites of infection and their The IncuCyte® Chemotaxis transendothelial migration assay is
subsequent migration through the endothelium are critical a fully automated solution to quantify directional leukocyte
steps of the immune response. This process of transendothelial migration across an endothelial monolayer using kinetic, image-
migration (TEM) is essential in order for leukocytes to respond based measurements. This label-free assay is suitable for testing
to foreign microorganisms, but if uncontrolled, can lead to multiple cell type pairings, evaluation of immunomodulation with
autoimmune disorders such as inflammatory bowel disease blocking antibodies or inhibitors, along with the associated ability
and rheumatoid arthritis. Assays to evaluate the extravasation to assess monolayer integrity throughout the assay.
of leukocytes are also essential for the study of host-defense
response and inflammatory disorders. The transendothelial migration assay is conducted in a 96-
well IncuCyte® ClearView Chemotaxis plate consisting of an
optically clear membrane insert and reservoir that allows for
In this chapter, we will review live-cell transendothelial cell direct visualization of leukocyte migration across an endothelial
migration assay, which accurately quantifies directional monolayer. The plate is placed in the IncuCyte® instrument, a
leukocyte migration across an endothelial monolayer in real fully automated live-cell analysis system with integrated image
time. This approach allows for the assay to be completed in a analysis tools, thus eliminating laborious endpoint analysis.
physiologically relevant environment and automatically analyzes Kinetic data of the 96-well plate is supported by images that
images, alleviating technically challenging and cumbersome allow for visualization of endothelial monolayer integrity and
quantification steps in traditional approaches. leukocyte diapedesis.

Shortcomings of Traditional Assays Live-Cell Imaging and Analysis Approaches

• Standard Boyden chamber surfaces are • Acquisition of high-definition, phase contrast images enable verification of intact
not easily amenable to imaging. endothelium throughout the experiment.
• Require use of intrinsically toxic dyes. • Eliminates use of intrinsically toxic dyes.
• Requires fixing, staining and cell • Fixing, staining and cell scraping steps not required.
scraping steps.
• Quantitate cells on top and bottom of the membrane to confirm movement.

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