Characterization of a novel angiogenic model based on stable, fluorescently labelled endothelial cell lines amenable to scale-up for high content screening.

Background: Blood vessel formation is important for many physiological and pathological processes and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumour or promoting 'normalization' of tumour vasculature in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows the investigation of cell-cell and cell-matrix interactions in a controlled environment and is therefore a crucial step in developing HCS (high content screening) and HTS (high throughput screening) assays to search for modulators of blood vessel formation.

Plasma membrane assays and three-compartment image cytometry for high content screening.

High throughput image cytometers analyze individual cells in digital photomicrographs by first assigning pixels within each image to plasma membrane, cytoplasm, nucleus, or other regions. In this study, we report on a novel algorithm that: 1) identifies plasma membrane regions to measure changes in plasma membrane-associated proteins (protein kinase C [PKC] alpha, N-cadherin, E-cadherin, vascular endothelium [VE]-cadherin, and pan-cadherin) that regulate cell division, migration, and adhesion and 2) delineates the cell for generalized three-compartment image cytometry. Validation assays were performed for these proteins on cells cultured in 96-well plates and also for tissue sections obtained from transgenic and chemical carcinogenic models of skin cancer.

Statistics of assay validation in high throughput cell imaging of nuclear factor kappaB nuclear translocation.

This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor kappaB (NF kappaB) in HeLa cells. We quantified cellular inhibition of cytoplasmic-nuclear translocation of NF kappaB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1alpha using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell.

Advances in molecular labeling, high throughput imaging and machine intelligence portend powerful functional cellular biochemistry tools.

Cellular behavior is complex. Successfully understanding systems at ever-increasing complexity is fundamental to advances in modern science and unraveling the functional details of cellular behavior is no exception. We present a collection of prospectives to provide a glimpse of the techniques that will aid in collecting, managing and utilizing information on complex cellular processes via molecular imaging tools.