JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.
1Department of Otolaryngology, University of California, San Francisco, 2Department of Chemistry, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Department of Pharmaceutical Chemistry, University of California, San Francisco, 5Tetrad Graduate Program, University of California, San Francisco, 6Center for Systems and Synthetic Biology, University of California, San Francisco, 7Chemistry and Chemical Biology Graduate Program, University of California, San Francisco
We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.
Published October 23, 2014. Keywords: Bioengineering, monovalent quantum dots, single particle tracking, SNAP tag, steric exclusion, phosphorothioate, DNA, nanoparticle bioconjugation, single molecule imaging
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 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
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 Computer Science and Engineering, University of California, Riverside, 2Department of Entomology, University of California, Riverside, 3Institute of Mathematics and Computer Sciences, University of São Paulo - USP, 4ISCA Technologies
We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.
Published October 15, 2014. Keywords: Bioengineering, flying insect detection, automatic insect classification, pseudo-acoustic optical sensors, Bayesian classification framework, flight sound, circadian rhythm
1Heart Research Center Goettingen, 2Clinic of Cardiology & Pulmonology, University Medical Center Goettingen, 3German Center for Cardiovascular Research (DZHK) partner site Goettingen, 4BioMET, Center for Biomedical Engineering & Technology, University of Maryland School of Medicine
In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.
Published October 15, 2014. Keywords: Bioengineering, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
1Center for Biotechnology Education, Johns Hopkins University, 2PBS Biotech, Inc.
Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.
Published October 10, 2014. Keywords: Bioengineering, single-use bioreactor, cell culture, mammalian cell culture, pneumatic wheel, upstream bioprocessing, Air-Wheel bioreactor
1NEST Laboratory, Scuola Normale Superiore, 2Center for Nanotechnology Innovation, Instituto Italiano di Tecnologia, 3Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine
Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.
Published October 9, 2014. Keywords: Bioengineering, fluorescence, protein dynamics, lipid dynamics, membrane heterogeneity, transient confinement, single molecule, GFP
1Institute of Immunology & Experimental Oncology, Center for Biomedical Education and Research (ZBAF), Witten/Herdecke University
Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.
Published October 5, 2014. Keywords: Bioengineering, cell migration, 3D collagen matrix, cell tracking
1NanoScience Technology Center, University of Central Florida
This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.
Published October 3, 2014. Keywords: Bioengineering, cantilever, in vitro, contraction, skeletal muscle, NMJ, cardiomyocytes, functional