Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.
Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.
Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.
We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.
1Laboratory of Applied Nutrition, School of Physical Education and Sport, University of Sao Paulo, 2Aerobic Performance Research Group, School of Physical Education and Sport, University of Sao Paulo, 3Laboratory of Neuromuscular Adaptations to Strength Training, School of Physical Education and Sport, University of Sao Paulo, 4Martial Arts and Combat Sports Research Group, School of Physical Education and Sport, University of Sao Paulo
This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.
Dry Oxidation and Vacuum Annealing Treatments for Tuning the Wetting Properties of Carbon Nanotube Arrays
This article describes a simple method to fabricate vertically aligned carbon nanotube arrays by CVD and to subsequently tune their wetting properties by exposing them to vacuum annealing or dry oxidation treatment.
Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.
Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model
1Department of Applied Physics and Physico-Informatics, Keio University, 2Department of Biochemistry, School of Medicine, Keio University, 3Exploratory Research for Advanced Technology (ERATO), Suematsu Gas Biology Project, Japan Science and Technology Agency (JST)
An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.
This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.
We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited.
The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.
1Department of Biomedical Engineering, The Ohio State University, 2Department of Biomedical Informatics, The Ohio State University, 3Comprehensive Wound Center, The Ohio State University, 4Department of Surgery, The Ohio State University
A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.
Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.
1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California
We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.
The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.
This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.
An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.
Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Center for Neural Development and Disease, University of Rochester Medical Center, 3Deptartment of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center
Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.
Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.
Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.
1Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 2Lab. PALM, Université de Caen Basse-Normandie, 3Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children's Hospital, Harvard Medical School, 4ISS, INC.
We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.
The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.
The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.
A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.
This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems
Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.
We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.
This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.
We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.
We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.
This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.
The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.
We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.
1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Department of Anesthesiology, Perioperative and Pain Medicine, Children’s Hospital Boston
Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.
Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.
1Buck Institute for Age Research, Novato, CA, 2Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham - UAB, 3Seahorse Bioscience, North Billerica, MA
A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.
Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)
We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).
Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.
Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling
A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.
Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.
We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.
This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.
Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.
In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model
1Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2Department of Radiology, University of Texas Southwestern Medical Center, 3Department of Radiation Oncology, Kyoto University Graduate School of Medicine
Bioluminescence imaging of hypoxia inducible factor-1α activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.
Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.
Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions
This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.
Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.
We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.
1Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 2Muscle Biology Research Group, University of Missouri-Kansas City, 3Pharmacology division, College of Pharmacy, DHLRI, Ohio State University
We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.
Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.