JoVE Clinical and Translational Medicine
1Division of Molecular Oncology, IRCCS, San Raffaele Scientific Institute, 2Department of Haemato-Oncology, King's College London, 3IFOM, FIRC Institute of Molecular Oncology, 4Università Vita-Salute San Raffaele
Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.
Published October 19, 2014. Keywords: Medicine, Lymphocytes, Chronic Lymphocytic Leukemia, 2D Electrophoresis, Mass Spectrometry, Cytoskeleton, Migration
1Department of Psychology, Rutgers University
Here we describe a touch-screen visual search paradigm that can be used to study threat detection across the lifespan. The paradigm has already been used in various studies demonstrating that both children and adults detect threatening stimuli like snakes, spiders, and angry faces faster than non-threatening stimuli.
Published October 19, 2014. Keywords: Behavior, Detection, threat, attention, attentional bias, anxiety, visual search
1Department of Neurosurgery, University Medical Center Utrecht, 2Brain Center Rudolf Magnus, University Medical Center Utrecht
Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.
Published October 19, 2014. Keywords: Bioengineering, exosomes, microvesicles, extracellular vesicles, quantification, characterization, Tunable Resistive Pulse Sensing, qNano
1Department of Microbiology and Immunology and Department of Pathology, Queen's University, 2Ask Science Products Inc.
This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.
Published October 18, 2014. Keywords: Molecular Biology, Electroporation, Indium-Tin oxide, signal transduction, gap junctional communication, peptides, Stat3
1Department of Biological Engineering, Massachusetts Institute of Technology, 2Environmental Toxicology, Chulabhorn Graduate Institute, 3Department of Biomedical Engineering, University of Minnesota
We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.
Published October 18, 2014. Keywords: Bioengineering, comet assay, electrophoresis, microarray, DNA damage, DNA repair
1School of Life Sciences, University of Nottingham
Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.
Published October 17, 2014. Keywords: Chemistry, Kinesin, ATP turnover, mantATP, mantADP, stopped-flow fluorescence, microtubules, enzyme kinetics, nucleotide
1Department of Neuroscience, Ohio State University
Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.
Published October 17, 2014. Keywords: Bioengineering, adhesion, aggregation, Fc-fusion, cadherin, protocadherin
1Department of Biochemistry, University of Leicester
The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.
Published October 16, 2014. Keywords: Biochemistry, structural biology, protein expression, recombinant protein, mammalian cell, transfection, polyethylenimine, suspension culture, affinity purification.
1Department of Biological Sciences, Rensselaer Polytechnic Institute
Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.
Published October 16, 2014. Keywords: Microbiology, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
JoVE Clinical and Translational Medicine
1Orthopaedic Hospital Research Center, Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 2PerkinElmer, 3Department of Dermatology, Johns Hopkins University School of Medicine, 4Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine
Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.
Published October 16, 2014. Keywords: Infection, imaging, optical, CT, bioluminescence, fluorescence, staphylococcus, infection, inflammation, bone, orthopaedic, implant, biofilm