JoVE Immunology and Infection
1Department of Pharmacology, University of Minnesota, 2Department of Surgery, University of Minnesota, 33M Corporate Research Laboratory
Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.
Published November 27, 2014. Keywords: Immunology, Fluorometry, phagocytosis, high throughput assay, actin polymerization, immunology
JoVE Immunology and Infection
1Department of Molecular and Biomedical Sciences, University of Maine, 2Graduate School of Biomedical Sciences and Engineering, University of Maine
In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.
Published November 27, 2014. Keywords: Immunology, Zebrafish, mucosal candidiasis, mucosal infection, epithelial barrier, epithelial cells, innate immunity, swimbladder, Candida albicans, in vivo.
JoVE Immunology and Infection
1Department of Microbiology & Immunology, The University of Texas Medical Branch, 2Department of Pathology, The University of Texas Medical Branch, 3Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, The University of Texas Medical Branch
Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.
Published November 27, 2014. Keywords: Immunology, West Nile Virus, Dendritic cells, T cells, cytokine, proliferation, in vitro
1Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA), 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles (UCLA)
We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.
Published November 26, 2014. Keywords: Stem Cell Biology, Human induced pluripotent stem cells, STEMCCA, factor-free, GMP, xeno-free, quantitative PCR
1Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine
We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.
Published November 26, 2014. Keywords: Genetics, Xist, X-chromosome inactivation, FISH, histone methylation, epigenetics, long non-coding RNA
1Department of Biotechnology, Delft University of Technology
The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.
Published November 26, 2014. Keywords: Biochemistry, Redox titration, electron paramagnetic resonance, Nar1, cofactor, iron-sulfur cluster, mononuclear iron, midpoint potential
1Center for Narcolepsy, Sleep and Health Research, University of Illinois at Chicago, 2Department of Pharmacology, University of Illinois at Chicago, 3Department of Biobehavioral Health Science, University of Illinois at Chicago
Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.
Published November 25, 2014. Keywords: Neuroscience, neuroscience, nodose ganglia, vagus nerve, EMG, serotonin, apnea, genioglossus, cannabinoids
1KY Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, 2Hotchkiss Brain Institute, Department of Clinical Neurosciences, University of Calgary
We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.
Published November 25, 2014. Keywords: Neuroscience, spinal cord injury, axon, myelin, two-photon excitation microscopy, Nile Red, axonal degeneration, axonal dieback, axonal retraction
1Emmy Noether Group, Institute of Zoology, University of Cologne
Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.
Published November 25, 2014. Keywords: Neurobiology, crustacean, dissection, extracellular recording, fictive locomotion, motor neurons, locomotion
JoVE Clinical and Translational Medicine
1Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Institute for Infectious Disease Research, McMaster University
Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.
Published November 24, 2014. Keywords: Virology, cotton rat, oncolytic virus, animal handling, bovine herpesvirus type 1