ABSTRACT
Flow cytometry (FCM) is an important technology with a broad spectrum of applications ranging from basic research to clinical diagnostics. In a typical FCM experiment, thousands of cells are queried with respect to size, shape, and abundance of multiple cell surface antigens. Recent advances in FCM techniques and instrumentation have enabled researchers to raise the throughput of experimentation dramatically. However, data analysis has remained a time-consuming activity requiring significant manual intervention for gating as well as for overall data reduction and interpretation. Presented in this article is a novel, algorithmically flexible, internally developed, software framework for the analysis of plate-based FCM data for high-throughput screening (HTS). Utilizing a post-treatment pooling strategy, >87,000 individual wells representing over 240,000 compounds were automatically gated, percent of control (POC) calculated, results assembled, deconvolved, and sorted, allowing researchers to visually assess wells of interest in minutes.
Subject(s)
Drug Evaluation, Preclinical/statistics & numerical data , Flow Cytometry/statistics & numerical data , Software , Algorithms , Cell Line, Tumor , Data Interpretation, Statistical , Drug Evaluation, Preclinical/instrumentation , Drug Evaluation, Preclinical/methods , Flow Cytometry/instrumentation , Flow Cytometry/methods , Humans , Small Molecule LibrariesABSTRACT
As technologies for high throughput and high content screening continue to evolve, new platforms for quantitative cellular imaging will play an increasingly important role in identifying and profiling lead compounds. To gain insight into the effects of a compound on cell morphology or intracellular events, it is necessary to have quality images and the ability to enumerate thousands of data points for statistical relevance. Imaging flow cytometry combines many of the features of flow cytometry, microscopy and imaging as well as a number of unique characteristics. The result is an instrument capable of highly quantitative analysis of cellular behaviors such as receptor internalization, phagocytosis, cell-cell communication, apoptosis and nuclear translocation. This promising new technology and unique type of flow cytometry provides enhanced capabilities for highly multiplexed assays. Here, we review the capabilities of the ImageStream imaging cytometer and discuss several applications relevant to compound screening and profiling.
Subject(s)
Drug Discovery/methods , Flow Cytometry/methods , High-Throughput Screening Assays/methods , Blood Cells/cytology , Blood Cells/drug effects , Drug Discovery/instrumentation , Flow Cytometry/instrumentation , High-Throughput Screening Assays/instrumentation , Humans , Pharmaceutical Preparations , SoftwareABSTRACT
Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for reducing excess hepatic glucose production in patients with type 2 diabetes. Despite extensive efforts, there is currently no human therapeutic that directly inhibits the glucagon/glucagon receptor pathway. We undertook a novel approach by generating high-affinity human monoclonal antibodies (mAbs) to the human glucagon receptor (GCGR) that display potent antagonistic activity in vitro and in vivo. A single injection of a lead antibody, mAb B, at 3 mg/kg, normalized blood glucose levels in ob/ob mice for 8 days. In addition, a single injection of mAb B dose-dependently lowered fasting blood glucose levels without inducing hypoglycemia and improved glucose tolerance in normal C57BL/6 mice. In normal cynomolgus monkeys, a single injection improved glucose tolerance while increasing glucagon and active glucagon-like peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.
Subject(s)
Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Glucose/metabolism , Homeostasis/drug effects , Receptors, Glucagon/antagonists & inhibitors , Animals , Blood Glucose/metabolism , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Endocytosis/drug effects , Flow Cytometry , Glucose Tolerance Test , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Kinetics , Ligands , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effectsABSTRACT
In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.
