Page 61 - Live-cellanalysis handbook

Kinetic Transendothelial Migration Assays

Sample Results

Assessment of monolayer integrity and visualization of TEM
Human umbilical vein endothelial cells (HUVECs) were grown multimeter. HUVEC monolayers were grown for 24 hours in EGM-
to confluence on top of fibronectin, a physiologically relevant 2, then growth medium was removed and 60μL of DPBS-/- was
basement membrane. Integrity of the monolayer was evaluated placed into the insert wells and 200 μL into the reservoir wells. The
both prior to leukocyte addition, using electrical resistance average resistance for inserts containing a monolayer was 31.9±1.7
measurements as well as staining for E-Selectin, and after compared to wells without cells14.9±0.9 (N=9 per condition),
leukocyte addition by assessing phase contrast images. indicative of monolayer formation over the insert pores (Figure
1B).
HUVECs were seeded at 6,000 cells per ClearView insert and
allowed to form a monolayer for approximately 24 hrs. Cells were Label-free CD3/CD28 Dynabead activated primary T cells were
permeabilized and fixed, and VE-cadherin adhesion junctions added to the endothelial monolayer cultured on fibronectin in
(green) were visualized from assembled 10x Z-stack confocal the presence or absence of migration modulators. Images were
images; dashed circles mark membrane pore locations (Figure 1A). acquired every minute (Figure 2) using the IncuCyte system’s 10x
objective. Yellow and orange arrows indicate leukocytes moving
Resistance measurements of HUVEC monolayers cultured on a between HUVEC cells and eventually down the pores (blue circles)
supporting matrix of fibronectin was measured using a digital of the ClearView insert.
A B

Figure 1. Assessment of monolayer integrity. VE-cadherin adhesion junctions (green) were visualized (A) and monolayer formation confirmed via
measurement of resistance across the inserts (B).

Figure 2. Visualization of leukocyte extravasation.

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