Este estudio fue financiado por los Institutos Canadienses de Investigación en Salud y Canadá Genoma. JS tiene premios salario apoyo de la Fundación del Patrimonio de Alberta para la Investigación Médica, Heart and Stroke Foundation de Canadá y la Asociación Canadiense de Diabetes. El laboratorio desea agradecer la asistencia técnica de los instrumentos de Oroboros durante la adquisición de la técnica de fibra de saponina-permeabilizadas.

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La técnica de saponina-permeabilized fibras cardiacas ofrece un compromiso único entre in vitro e in vivo en la evaluación del consumo de oxígeno mitocondrial OXPHOS. Ventajas de esta técnica incluyen el aumento de relevancia fisiológica en comparación con las mitocondrias aisladas como la arquitectura celular se conserva. Mientras que la membrana plasmática se degrada, las estructuras intracelulares de membrana como la mitocondria 12, 14, 14 del retículo sarcoplásmico, miofilament...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>100% Ethanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>HC600</td>
<td>&nbsp;</td>
<tr>
<td>70% Ethanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>HC-1000</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-diphosphate monopotassium salt dihydrate (ADP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5285</td>
<td>&nbsp;</td>
<tr>
<td>Albumin from bovine serum essentially fatty acid&#x2013;free</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-6003</td>
<td>&nbsp;</td>
<tr>
<td>Antimycin A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8674</td>
<td>&nbsp;</td>
<tr>
<td>Ascobic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4403</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-triphosphate disodium salt hydrate (ATP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2383</td>
<td>&nbsp;</td>
<tr>
<td>Atractyloside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6882</td>
<td>&nbsp;</td>
<tr>
<td>Calcium carbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4830</td>
<td>&nbsp;</td>
<tr>
<td>Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2920</td>
<td>&nbsp;</td>
<tr>
<td>Cytochrome c</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7752</td>
<td>&nbsp;</td>
<tr>
<td>Digitonin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D141</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D9779</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene glycol-bis-(2-aminoethylether)-N,N,N&#x2032;,N&#x2032;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E4378</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>27647</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I5513</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Pfizer</td>
<td>&nbsp;</td>
<td>Ketaset</td>
</tr>
<tr>
<td>Lactobionic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>153516</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9272</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride hexahydrate (MgCl2&#x2219;6H2O)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9272</td>
<td>&nbsp;</td>
<tr>
<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M1000</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M3671</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N&#x2019;,N&#x2019;-Tetramethyl- pphenylenediamine Dihydrochloride (TMPD)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T3134</td>
<td>&nbsp;</td>
<tr>
<td>Oligomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O4876</td>
<td>&nbsp;</td>
<tr>
<td>Phosphocreatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7936</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Hydroxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5958</td>
<td>&nbsp;</td>
<tr>
<td>Potassium cyanide</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>60178</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5655</td>
<td>&nbsp;</td>
<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R8875</td>
<td>&nbsp;</td>
<tr>
<td>Saponin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>47036</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pentobarbital</td>
<td>&nbsp;</td>
<td>Ceva Sante Animale</td>
<td>1715 138</td>
<td>Conc. 54.7 mg/ml</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>Succinic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3674</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7903</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8691</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Bayer</td>
<td>&nbsp;</td>
<td>Rompun</td>
</tr>
<tr>
<td>ddH2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ice</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Oroboros Oxygraph-2k</td>
<td>&nbsp;</td>
<td>Oroboros Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>21905-026</td>
<td>&nbsp;</td>
<tr>
<td>15ml polypropylene centrifuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89004-368</td>
<td>&nbsp;</td>
<tr>
<td>50ml polypropylene centrifuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89004-364</td>
<td>&nbsp;</td>
<tr>
<td>Straight Jewelers Forceps</td>
<td>&nbsp;</td>
<td>George Tiemann &amp; Co.</td>
<td>160-50B</td>
<td>&nbsp;</td>
<tr>
<td>Curved Jewelers Forceps</td>
<td>&nbsp;</td>
<td>George Tiemann &amp; Co.</td>
<td>160-57B</td>
<td>&nbsp;</td>
<tr>
<td>Straight Surgery Scissors</td>
<td>&nbsp;</td>
<td>George Tiemann &amp; Co.</td>
<td>105-402</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Surgical Blade</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>BD371610</td>
<td>&nbsp;</td>
<tr>
<td>0.45-&mu;m Syringe filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA28145-485</td>
<td>&nbsp;</td>
<tr>
<td>pH meter</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA11388-308</td>
<td>&nbsp;</td>
<tr>
<td>Glass Petri dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-300</td>
<td>&nbsp;</td>
<tr>
<td>12-well Polystyrene Tissue Culture Plates</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82050-926</td>
<td>&nbsp;</td>
<tr>
<td>Plate Stirrer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>97042-594</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Microbars</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-511-67</td>
<td>&nbsp;</td>
<tr>
<td>Weigh Scale</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA11278-162</td>
<td>&nbsp;</td>
<tr>
<td>10&mu;l Hamilton Micro Syringe</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-815-1</td>
<td>&nbsp;</td>
<tr>
<td>25&#x03BC;l Hamilton Micro Syringe</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-824-7</td>
<td>&nbsp;</td>
<tr>
<td>50&mu;l Hamilton Micro Syringe</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-824-5</td>
<td>&nbsp;</td>
<tr>
<td>Nalgene Squeeze Bottles</td>
<td>&nbsp;</td>
<td>Wilkem Scientific</td>
<td>LNA2407-1000</td>
<td>&nbsp;</td>
<tr>
<td>Polystyrene Weighing Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89106-750</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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respirometric oxidative phosphorylation assessment in saponin-permeabilized cardiac fibers

Investigación de la función mitocondrial representa un parámetro importante de la fisiología cardiaca como las mitocondrias están implicadas en el metabolismo energético, el estrés oxidativo, la apoptosis, el envejecimiento, encefalomiopatías mitocondrial y la toxicidad del fármaco. Teniendo en cuenta esto, las tecnologías para medir la función mitocondrial cardiaco están en demanda. Una técnica que emplea un enfoque integral para medir la función mitocondrial es respirométrica fosforilación oxidativa (OXPHOS) análisis. El principio de la evaluación de la fosforilación oxidativa respirometría se centra en medir la concentración de oxígeno que utiliza un electrodo de Clark. A medida que el haz de fibras permeabilized consume el oxígeno, la concentración de oxígeno en la disminución de cámara cerrada. Utilizando sustrato seleccionado desacoplador inhibidor de los protocolos de valoración, los electrones se proporcionan a sitios específicos de la cadena de transporte de electrones, lo que permite la evaluación de la función mitocondrial. Técnicas de preparación antes del análisis de respirometría de la función mitocondrial, mecánicos y químicos se utilizan para permeabilizar el sarcolema de las fibras musculares. Permeabilización química emplea saponina para perforar de forma selectiva la membrana celular, manteniendo la arquitectura celular. Este documento describe minuciosamente los pasos a seguir en la preparación de saponina de piel fibras cardiacas para medir el consumo de oxígeno para evaluar la fosforilación oxidativa mitocondrial. Además, la solución de problemas asesoramiento, así como sustratos específicos, los inhibidores y desacopladores de que se puede utilizar para determinar el funcionamiento de las mitocondrias en sitios específicos de la cadena de transporte electrónico se proporcionan. Es importante destacar que el protocolo descrito puede aplicarse fácilmente en el tejido cardíaco y esquelético de varios modelos animales y muestras humanas.

Watch this Scientific Journal Video about Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers at JoVE.com

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Saponina permeabilized preparación de la fibra junto con el análisis de la fosforilación oxidativa respirométrica proporciona una evaluación integradora de la función mitocondrial. Respiración mitocondrial en los estados fisiológicos y patológicos pueden reflejar diferentes influencias regulatorias, incluidas las interacciones mitocondrial, la morfología y la bioquímica.

Evaluación de respirometría la fosforilación oxidativa en saponina-permeabilized fibras cardíacas

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

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24.6K vistas.  University of Calgary. Saponina permeabilized preparación de la fibra junto con el análisis de la fosforilación oxidativa respirométrica proporciona una evaluación integradora de la función mitocondrial. Respiración mitocondrial en los estados fisiológicos y patológicos pueden reflejar diferentes influencias regulatorias, incluidas las interacciones mitocondrial, la morfología y la bioquímica.

Video: Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Investigación

Biología

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioingeniería Humanos redes microvasculares en ratones inmunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital de la microcirculación en el músculo de ratón Cremaster para el análisis de la migración de la célula madre periféricas

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Evaluación de calcio en Sparks intactas las fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Analysis of Oxidative Stress in Zebrafish Embryos

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 &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; 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.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Análisis de estrés oxidativo en embriones de pez cebra

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA para identificar ubiquitina y reguladores del sistema ubiquitina-como de las vías biológicas en células de mamífero en cultivo

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

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.

Método para la Evaluación de los efectos de un rango de longitudes de onda e intensidades de / Terapia de luz del infrarrojo cercano Red sobre el estrés oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Medición de la resistencia de estrés oxidativo<em&gt; Caenorhabditis elegans</em&gt; 96 pocillos en placas de microtitulación

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Evaluación de los miofilamentos Ca<sup&gt; 2+</sup&gt; Sensibilidad cardíaca subyacente excitación-contracción de acoplamiento

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Análisis de la red α-Actinin en cardiomiocitos humanos derivados de iPSC mediante microscopía de localización de molécula única

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...