Page 62 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Real-time kinetic quantitation of label-free TEM
CD3/CD28 Dynabead activated primary T-cells were seeded on a processing separates T cells (outlined in yellow) from the HUVEC
HUVEC monolayer cultured on fibronectin in IncuCyte ClearView monolayer (Figure 3B). Images are processed as they are acquired,
chemotaxis plates. Live cell images were captured at regular and data can be plotted in real time as a decrease in area on the
time intervals, and directed transendothelial migration toward top side of the membrane for leukocytes that extravasate the
CXCL12 (SDF-1a) was quantified using image analysis algorithms. endothelial monolayer and down the pore.
Significant transendothelial migration, or diapedesis, was observed
in response to increasing concentrations of SDF-1a, and could The insert containing T cell:HUVEC monolayer co-cultures was
be completely inhibited using high concentrations of AMD3100, exposed to 3-fold decreasing concentrations of CXCL12 (SDF-1a)
a selective inhibitor of CXCR4 and CXCL12 (SDF-1a) mediated (Figure 4A). Images were acquired every 30 minutes and phase
chemotaxis. analysis was performed. Analysis of pharmacological response was
performed at t=6hr; each data point represents mean ±SEM, N=4
Whole-well, phase-contrast images represent the top-side of the (Figure 4B).
membrane at t=0 hour time point (Figure 3A). Automated image

A B

Figure 3. Data quantification and analysis. Whole-well, phase-contrast images represent the top-side
of the membrane at t=0 hour time point (A). Automated image processing separates T cells (outlined
in yellow) from the HUVEC monolayer (B).

A B

Figure 4. Primary T cells extravasation toward CXCL12. CXCL12 (SDF-1a) induced TEM (A). Pharmacological response of T cell migration (B).

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