Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.
25 Related JoVE Articles!
Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry
Institutions: Karolinska Institutet, National Institute on Drug Abuse (NIDA).
Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2
. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3
. A wealth of evidence has been compiled in support of this theory 1,4
, an example being the mtDNA mutator mouse 5
; however, the precise role of mtDNA damage in aging is not entirely understood 6,7
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c
oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2
. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9
. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12
. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14
. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16
has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23
. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22
. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22
. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24
. Cytochrome c
and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9
. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22
. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25
. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.
Cellular Biology, Issue 57, aging, brain, COX/SDH, histochemistry, mitochondria, mitochondrial disease, mitochondrial dysfunction, mtDNA, mtDNA mutations, respiratory chain
Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT
Institutions: The Methodist Hospital Research Institute, Houston, The Methodist Hospital Research Institute, Houston.
Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1
. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via β adrenergic receptor 2, 3
. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6
. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8
. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13
, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18
F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of β-adrenoceptor antagonist propranolol 14, 15
, or the enhanced BAT activation by β3 agonist BRL37344 16
. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.
Molecular Biology, Issue 69, Neuroscience, Anatomy, Physiology, Medicine, Brown adipose tissue, mice, 18F-Fluorodeoxyglucose, micro-PET, PET, CT, CT scan, tomography, imaging
Localization, Identification, and Excision of Murine Adipose Depots
Institutions: University of Cincinnati College of Medicine.
Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro
experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.
Medicine, Issue 94, adipose, surgical, excision, subcutaneous adipose tissue (SQ), perivascular adipose tissue (PVAT), visceral adipose tissue (VAT), brown adipose tissue (BAT), white adipose tissue (WAT)
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Institutions: Loyola University Chicago.
Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria.
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2
DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2
DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2
. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.
Neuroscience, Issue 51, Mitochondrial membrane potential, reactive oxygen species, neuroscience, cortical neurons
Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers
Institutions: University of Calgary, University of Calgary.
Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.
Physiology, Issue 48, cardiac fibers, mitochondria, oxygen consumption, mouse, methodology
Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates
Institutions: Cytori Therapeutics Inc, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA.
In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.
Cellular Biology, Issue 79, Adipose Tissue, Stem Cells, Humans, Cell Biology, biology (general), enzymatic digestion, collagenase, cell isolation, Stromal Vascular Fraction (SVF), Adipose-derived Stem Cells, ASCs, lipoaspirate, liposuction
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response
Institutions: University of Alabama.
Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster.
Here, we have used the conserved innate immune response in Drosophila
to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster,
and this assay can be modified to be applicable to other Drosophila
tissues or even other small, whole-organism inflammation models.
Immunology, Issue 82, biology (general), environmental effects (biological, animal and plant), immunology, animal models, Immune System Diseases, Pathological Conditions, Signs and Symptoms, Life Sciences (General), Neuroinflammation, inflammation, nitric oxide, nitric oxide synthase, Drosophila, neurodegeneration, brain, Griess assay, nitrite detection, innate immunity, Parkinson disease, tissue culture
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue
Institutions: Massachusetts General Hospital/Harvard Medical School, China Pharmaceutical University, Perkin Elmer.
Brown adipose tissue (BAT), widely known as a “good fat” plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18
F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18
F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18
F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.
Medicine, Issue 92, Cerenkov luminescence imaging, brown adipose tissue, 18F-FDG, optical imaging, in vivo imaging, spectral unmixing
Viability Assays for Cells in Culture
Institutions: Duquesne University.
Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.
Cellular Biology, Issue 83, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells
Institutions: University of California, Los Angeles.
Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.
Cell Biology, Issue 16, human embryonic stem cells, mitochondria, oxidative phosphorylation, respiration, electron transport chain, native gel electrophoresis
Bioenergetic Profile Experiment using C2C12 Myoblast Cells
Institutions: Novato, CA, University of Alabama at Birmingham - UAB, North Billerica, MA.
The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2
into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.
Cellular Biology, Issue 46, Mitochondrial dysfunction, cellular, bioenergetics, metabolism, cancer, obesity, diabetes, aging, neurodegeneration
Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Institutions: University of Arkansas for Medical Sciences .
The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro
model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo
. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0
-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0
-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro
Cellular Biology, Issue 71, Biochemistry, Molecular Biology, Genetics, Pharmacology, Physiology, Medicine, Protein, Mitochondrial dysfunction, mitochondria, protein kinase C, renal proximal tubular cells, reactive oxygen species, oxygen consumption, electron transport chain, respiratory complexes, ATP, adenovirus, primary culture, ischemia, cells, flow cytometry
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets
Institutions: University of Alabama at Birmingham.
Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
Immunology, Issue 85, bioenergetics, translational, mitochondria, oxidative stress, reserve capacity, leukocytes
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Methods to Assess Subcellular Compartments of Muscle in C. elegans
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans
is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans
is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo
. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo
. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans
provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo
in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells
Institutions: Columbia University, Columbia University.
Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the Fo
-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
Bioengineering, Issue 77, Microbiology, Cellular Biology, Molecular Biology, Biochemistry, life sciences, roGFP, redox-sensitive green fluorescent protein, GO-ATeam, ATP, FRET, ROS, mitochondria, biosensors, GFP, ImageJ, microscopy, confocal microscopy, cell, imaging
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Institutions: Ben-Gurion University of the Negev.
The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro
, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans
. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo
, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.
Biochemistry, Issue 82, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice
Institutions: Hunter College, CUNY.
Efficient expression of transgenes in vivo
is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).
Genetics, Issue 87, hydrodynamic gene delivery, hydrodynamics-based transfection, mouse, gene therapy, plasmid DNA, transient gene expression, tail vein injection
Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles
Institutions: UMDNJ-Robert Wood Johnson Medical School, University of Missouri-Kansas City, Ohio State University .
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+
handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+
signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e.
, mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro
muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.
Physiology, Issue 69, extensor digitorum longus, soleus, in vitro contractility, calcium signaling, muscle-tendon complex, mechanic alternans
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
Institutions: University of Washington, Seattle.
The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1
, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+
, bioenergetics and redox signaling 3
mtPTP opening is triggered by matrix Ca2+
but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4
. In vitro
, mtPTP opening can be achieved by increasing Ca2+
concentration inside the mitochondrial matrix through exogenous additions of Ca2+
(calcium retention capacity). When Ca2+
levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+
release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+
handling (uptake and release, as measured by Ca2+
concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+
measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+
, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.
Cellular Biology, Issue 67, Mitochondria, respiration, mitochondrial permeability transition pore (mPTP), membrane potential, swelling, calcium, spectrofluorometer
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
Light/dark Transition Test for Mice
Institutions: Graduate School of Medicine, Kyoto University.
Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.
Neuroscience, Issue 1, knockout mice, transgenic mice, behavioral test, phenotyping