Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.
Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model
1The Markey Cancer Center, University of Kentucky College of Medicine, 2Graduate Center for Toxicology, University of Kentucky College of Medicine, 3Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, 4Department of Pediatrics, University of Kentucky College of Medicine
Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.
Published September 7, 2013. Keywords: Medicine, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.
Published November 1, 2013. Keywords: Immunology, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.
Published May 22, 2012. Keywords: Basic Protocols, PCR, optimization, primer design, melting temperature, Tm, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics
A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome
1Université Claude Bernard Lyon, 2Centre de Génétique et de Physiologie Moléculaires et Cellulaires, CNRS UMR 5534
This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
Published June 13, 2011. Keywords: Neuroscience, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution
1Institute of Biophysics, National Research Council of Italy, 2Dipartimento di Sistemi e Informatica, Università di Firenze, 3Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia
We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.
Published May 27, 2013. Keywords: Bioengineering, Biophysics, Neuroscience, Cellular Biology, Biomedical Engineering, Engineering (General), Life Sciences (General), Physics (General), Axon, tension release, Laser dissector, optical tweezers, force spectroscopy, neurons, neurites, cytoskeleton, adhesion, cell culture, microscopy
1Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio, 2Department of Cellular and Structural Biology, UT Health Science Center at San Antonio, 3Department of Pathology, UT Health Science Center at San Antonio, 4Department of Microbiology, UT Health Science Center at San Antonio, 5Cancer Therapy and Research Center, UT Health Science Center at San Antonio
Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model. The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants spontaneously arisen or after exposure to genotoxins.
Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.
Published July 23, 2013. Keywords: Plant Biology, Genetics, Molecular Biology, Cellular Biology, Virology, Microbiology, Bioengineering, Plant Viruses, Antibodies, Monoclonal, Green Fluorescent Proteins, Plant Proteins, Recombinant Proteins, Vaccines, Synthetic, Virus-Like Particle, Gene Transfer Techniques, Gene Expression, Agroinfiltration, plant infiltration, plant-made pharmaceuticals, syringe agroinfiltration, vacuum agroinfiltration, monoclonal antibody, Agrobacterium tumefaciens, Nicotiana benthamiana, GFP, DsRed, geminiviral vectors, imaging, plant model
1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Life Sciences Institute, University of Michigan, 4Department of Cell and Developmental Biology, University of Michigan, 5Department of Mechanical Engineering, University of Michigan
Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.