1Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute, 2Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University
Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.
Published March 25, 2015. Keywords: Molecular Biology, protein delivery, cell-penetrating peptide, zinc-finger, protein transduction domain, chemical biology, molecular biology
1Department of Cell and Molecular Physiology, Loyola University Chicago, 2Department of Biomedical Engineering, University of Alabama at Birmingham, 3Department of Bioengineering, Clemson University
This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.
Published March 23, 2015. Keywords: Cellular Biology, Cellular optical mapping, cultured atrial monolayer, action potential, calcium transient, voltage-sensitive dye, calcium dye
1Department of Biotechnology, University of Verona, Verona, Italy, 2Department of Internal Medicine, University of Perugia, Perugia, Italy
In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.
Published March 23, 2015. Keywords: Plant Biology, Plant biotechnology, transient expression, stable expression, transgenic plant, Nicotiana tabacum, Nicotiana benthamiana, Baculovirus/insect cells, recombinant protein
1Chemistry Department, Elon University, 2Department of Neurobiology and Anatomy, Wake Forest School of Medicine, 3Neuroscience Graduate Program, Wake Forest School of Medicine, 4ALS Center Translational Science Unit, Wake Forest School of Medicine
Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.
Published March 23, 2015. Keywords: Cellular Biology, Mitochondria, TMRE, cytokines, ALS, HEK cells, fluorescence, mitochondrial dysfunction, mitochondrial membrane potential, cytochrome c
1Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, 2School of Animal Biology and The Oceans Institute, The University of Western Australia, 3Experimental and Regenerative Neurosciences, School of Anatomy, Physiology and Human Biology, The University of Western Australia
Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.
Published March 21, 2015. Keywords: Engineering, Red light therapy, reactive oxygen species, oxidative stress, photobiomodulation, optimization, irradiation
1Blood Systems Research Institute, 2Department of Medicine, University of California, San Francisco, 3Department of Laboratory Medicine, University of California, San Francisco
Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.
Published March 17, 2015. Keywords: Cellular Biology, microvesicles, flow cytometry, exosomes, extracellular vesicles, high throughput, microparticles
1Carl Hayden Bee Research Center, USDA-ARS, 2Coupeville, WA, USA, 3Ecotoxicology Division, Eurofins Agroscience Services, Inc.
Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.
Published March 17, 2015. Keywords: Molecular Biology, pollen, nutrition, microbes, protein, amino acids, Africanized bees, genotype, Apis mellifera, scutellata
1Discipline of Physiology, University of Sydney, 2Discipline of Biomedical Science, University of Sydney
Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.
Published March 14, 2015. Keywords: Molecular Biology, Vestibular, mitochondria, infrared, neuroprotection, aging, oxidative stress
1Institute of Virology, National Reference Centre for Hepatitis C, Essen University Hospital, University of Duisburg-Essen
The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.
Published March 13, 2015. Keywords: Molecular Biology, Dried blood spots, filter paper cards, specimen storage, infectious diseases, hepatitis B virus, hepatitis C virus, human immunodeficiency virus
1Forensic Science Graduate Program, Biochemistry Track, University of Central Florida, 2Department of Chemistry, University of Central Florida, 3National Center for Forensic Science, University of Central Florida
Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in ‘touch DNA’ samples, together with an enhanced amplification protocol involving a one-step 5 µl micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).
Published March 9, 2015. Keywords: Basic Protocol, Forensic Science, Touch DNA Evidence, Micro-manipulation, Cell Isolation and Recovery, DNA Profiling, Short Tandem Repeat (STR) Analysis