Improved visualization and quantitative analysis of drug effects using micropatterned cells.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Capripoxvirus G-protein-coupled chemokine receptor: a host-range gene suitable for virus animal origin discrimination.

The genus Capripoxvirus within the family Poxviridae comprises three closely related viruses, namely goat pox, sheep pox and lumpy skin disease viruses. This nomenclature is based on the animal species from which the virus was first isolated, respectively, goat, sheep and cattle. Since capripoxviruses are serologically identical, their specific identification relies exclusively on the use of molecular tools. We describe here the suitability of the G-protein-coupled chemokine receptor (GPCR) gene for use in host-range grouping of capripoxviruses. The analysis of 58 capripoxviruses showed three tight genetic clusters consisting of goat pox, sheep pox and lumpy skin disease viruses. However, a few discrepancies exist with the classical virus-host origin nomenclature: a virus isolated from sheep is grouped in the goat poxvirus clade and vice versa. Intra-group diversity was further observed for the goat pox and lumpy skin disease virus isolates. Despite the presence of nine vaccine strains, no genetic determinants of virulence were identified on the GPCR gene. For sheep poxviruses, the addition or deletion of 21 nucleic acids (7 aa) was consistently observed in the 5' terminal part of the gene. Specific signatures for each cluster were also identified. Prediction of the capripoxvirus GPCR topology, and its comparison with other known mammalian GPCRs and viral homologues, revealed not only a classical GPCR profile in the last three-quarters of the protein but also unique features such as a longer N-terminal end with a proximal hydrophobic alpha-helix and a shorter serine-rich C-tail.