Enabling High Throughput
Larry Sklar, Ph.D., director, University of New Mexico Center for Molecular Discovery, pursues research on the application of flow cytometry for drug discovery and explores the molecular interactions at cell surfaces that lead to intracellular signaling. His laboratory is a member of the Molecular Libraries Program of the NIH Roadmap Initiative.
In his presentation, Dr. Sklar described his group’s use of a 384-well plate-based HyperCyt® high-content flow cytometry screening system from IntelliCyt. In development is a 1,536-well system capable of generating 250,000–500,000 data points/day. Their research focuses on the regulation of integrin, an adhesion molecular present on the surface of white blood cells.
Dr. Sklar and his team are conducting high-throughput, high-content screens to evaluate the ability of small molecules and repurposed drugs to activate signaling pathways that lead to changes in the conformation and affinity of integrins.
Dr. Sklar said that one of the most powerful aspects of flow cytometry for these types of studies was the ability to use a laser beam to generate a signal from a fluorescently labeled ligand that interacts with integrins on the leukocyte surface and detect in real-time associated changes in integrin affinity and cell activation that enable the leukocyte to interact with an endothelial cell. This technique enables studies of association/dissociation kinetics and the ability to experiment with interventions intended to modulate the dissociation rate.
The researchers have also developed fluorescence resonance energy transfer (FRET)-based assays to detect integrin activation and extension, using the data to correlate changes with agonist stimulation of various GPCRs, and apply the information gained on differential integrin regulation to pathway analysis. This knowledge could then feed back into the design of drug screens to identify small molecules that activate targeted pathways.