Page 63 - Live-cellanalysis handbook

Kinetic Transendothelial Migration Assays

Inhibition of leukocyte extravasation
For AMD3100 (CXCR4 receptor antagonist) and BIRT377 (allosteric were then plated at a density of 5,000 cells per well on to a HUVEC
modulator of LFA- 1) inhibition studies, CD3/CD28 activated monolayer then exposed to 100nM SDF-1a (EC80concentration).
T-cells were pre-treated for 1 hour at 37°C at indicated inhibitor Images were collected every hour over a 12 hour period and phase
concentrations, then seeded onto HUVEC monolayers and exposed analysis performed.
to 100nM SDF-1a (Figure 5A and C).
The observed increase in total cell area is due to proliferation of
Analysis of AMD3100 pharmacological response was performed at C3/CD28 activated T cells. The inability to fully inhibit TEM via
t=6 hr. Each data point represents mean ± SEM, N=4 (Figure 5B). BIRT 377 and neutralizing ICAM-1 is believed to be caused by the
For antibody inhibition, HUVEC monolayers cultured overnight centrifugation step, bringing the T cells to the HUVEC monolayer,
on fibronectin were ± pre-treated with neutralizing ICAM-1 for thus bypassing the cell adhesion step.
1 hour at 37°C (Figure 5D). CD3/CD28 Dynabead activated T cells

A B

C D

Figure 5. Treatment effects on leukocyte transendothelial migration. Inhibition of CXCL12 (SDF-1a) driven T cell migration by AMD3100 (A), BIRT 377
(C) and neutralizing ICAM-1. Analysis of AMD3100 pharmacological response (B). Migration across the endothelial layer was at least partially dependent
on ICAM-1, as neutralizing ICAM-1 antibodies, and treatments with an allosteric inhibitor of LFA-1, BIRT377, significantly inhibited SDF-1a mediated
diapedesis of T cells.

Conclusions

The IncuCyte® chemotaxis transendothelial assay can be used for real-time visualization
and automated analysis of transendothelial migration in a 96-well format, in real-time.

• Acquisition of high-definition, phase contrast images ensure endothelium integrity
throughout the experiment.
• Measurement of leukocyte extravasation is automatically processed without use of
intrinsically toxic dyes, fixing, staining and cell scraping steps.
• All data points can be validated by individual images or time-lapse movies to confirm
processing metrics, significantly enhancing the confidence in the measured response.

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