JoVE Immunology and Infection
1Center for Molecular Bacteriology and Infection, Imperial College London
The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.
1Center for Biotechnology Education, Krieger School of Arts and Sciences, Johns Hopkins University, 2Qiagen Sciences, Inc.
Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.
JoVE Clinical and Translational Medicine
1Department of Medicine, Northwestern University, 2Robert H. Lurie Cancer Center, Northwestern University, 3Center for Molecular Innovation and Drug Discovery, Northwestern University
This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.
1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology - Campus North, 2Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus North, 3Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus South
We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.
1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Program in Molecular Medicine, University of Maryland School of Medicine, 3Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, 4Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine, 5Department of Pathology and Biochemistry & Molecular Biology, University of Maryland School of Medicine
We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.
1Section of Molecular Genetics and Microbiology, The University of Texas at Austin, 2Department of Chemistry and Biochemistry, The University of Texas at Austin, 3The Institute of Cellular and Molecular Biology, The University of Texas at Austin
Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.
1Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London
Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.
JoVE Immunology and Infection
1Department of Immunotherapeutics (Medinet), University of Tokyo Hospital, 2MEDINET Co., Ltd
A method to expand γδ T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived γδ T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of γδ T cells can be applied to autologous cellular immunotherapy of cancer.
1Departments of Chemistry and Materials Science and Engineering, University of Utah
This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.
1Department of Genetics and Biochemistry, Clemson University
A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.