Computational protocol: Four-Dimensional Characterization of Thrombosis in a Live-Cell, Shear-Flow Assay: Development and Application to Xenotransplantation

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Protocol publication

[…] The sequential source images were stacked for their respective channels and analyzed using a component of Montage (Fluxion Biosciences) based on the Metamorph engine (Molecular Devices, Sunnyvale). To capture the most biologically and physiologically significant event within each channel, the signal threshold for each stack was determined from the image with the highest gross thrombosis. Thresholds were highly consistent within and between experiments. Fluorescent intensity (in arbitrary units, a.u.) and surface area coverage (SA, percent) and were extracted from image stacks and exported for data analysis.As fluorescence is a function of the labeled platelet mass, it was used as a measure of total thrombus volume (TV, in arbitrary units).[] SA was applied directly as a measure of adhesion in the x and y dimensions between the endothelium and perfused platelets.[] The fluorescence:SA ratio (FR, a.u.) determined relative binding in the z dimension, and was used as an index of platelet aggregation.[] Binding kinetics were measured by the time to 50% peak SA (T50, minutes). Mean peak TV, SA, FR and T50 values were compared by unpaired Student’s t-test. To correct for the well-described artifact of photobleaching, when analyzing serial time points a constant total fluorescence value was manually maintained for subsequent frames if fluorescence became degraded in the setting of visibly unchanged thrombus volume. Results were qualitatively assessed by three-dimensional surface renditions using ImageJ (National Institutes of Health, Bethesda).[] […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Blood Platelet Disorders, Thrombosis, Machado-Joseph Disease