Name: preparation of highly coupled rat heart mitochondria
Uploaded: 2010-09-23T17:30:00
Duration: 12 min 28 s
Description: Watch this Scientific Journal Video about Preparation of Highly Coupled Rat Heart Mitochondria at JoVE.com

We thank Prof. A. Vinogradov and Dr Vera Grivennikova for introduction of the authors to the field of mitochondrial biology and many useful insights and advice in this procedure. The authors are grateful to Mrs Amanda McKintock from the Queens University media office for the help with the video preparation.

Introduction
Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism. They can be obtained quickly and reliably in great quantity from wild type or knock-out animals. The general procedure consists of tissue digestion by trypsin, fractionation and differential centrifugation.
There are several conditions that have to be kept during Isolation of mitochondria from various tissues, including the heart (Fig. 1).
<ul>
 <li>The tissue has to be thoroughly minced, since it directly affects the yield of obtained mitochondria. Heart tissue is best to mince with small curved scissors and can be followed by use a Latapie tissue grinder or, if a tissue grinder is not available, a garlic press with outlet holes diameter of around 1.0 mm diameter.</li>
 <li>It is necessary to maintain the temperature of solutions at each step. Therefore the entire procedure has to be carried out in a cold room and all instruments and solutions have to be prepared and cooled in advance. If the temperature conditions are not kept stable several properties of the preparation can be lost. Mitochondrial structure is very sensitive to freezing so overfreezing of the suspension (for example during centrifugation) is not permitted, especially if studies of active transport are important.</li>
 <li>An important parameter is the time-length of the procedure; the isolation should be performed as quickly as possible and the obtained preparation has to be used immediately. Therefore, surgical instruments, solutions and appliances have to be prepared in advance.</li>
</ul>
Materials and instruments
Instruments
<ol>
 <li> Straight operating scissors, sharp point</li>
 <li> Curved scissors</li>
 <li> Forceps</li>
 <li> Petri dish</li>
 <li> Small funnel + piece of nylon filter (prewashed)</li>
 <li> 6 beakers 50 ml</li>
 <li> Two magnetic stirrers and two ice baths</li>
 <li> One big ice bath</li>
 <li> 2 small magnetic bars</li>
 <li> Spatulas for pellet scraping</li>
 <li> Centrifuge tubes</li>
 <li> Latapie tissue press (can be substituted with garlic press)</li>
 <li> Teflon-glass homogenisers 20ml and 1-2ml</li>
 <li> Waste beaker</li>
 <li> Eppendorf tubes for final mitochondrial suspension and samples for protein determination</li>
 <li> Ice baths</li>
</ol>
Solutions (calculated per 2 rats):
<ol>
 <li> Washing buffer: 0.3 M Sucrose, 10 mM HEPES (pH=7.2), 0.2 mM EDTA (1000 ml)</li>
 <li> Isolation buffer: 0.3 M Sucrose, 10 mM HEPES (pH=7.4), 0.2 mM EDTA, 1 mg/ml BSA (Sigma A6003) (100ml prepared from the Washing buffer). Bovine serum albumin can be substituted by less expensive fatty acid-free analogues. The same buffer or its isotonic variations can be used as Resuspending buffer.</li>
 <li> Trypsin (Sigma T8003) solution: 2.5mg/ml in 1mM HCl (0.5 ml)</li>
 <li> Trypsin inhibitor from soy bean (Sigma T9128) 6.5 mg/10 ml of Isolation buffer</li>
</ol>
Protocol
The general scheme of the procedure is shown on Fig. 1. Hearts should be cooled and washed in Washing buffer, ventricular tissue excised, minced and homogenised during photolytic treatment with trypsin. The suspension is centrifuged at low speed and the obtained supernatant is centrifuged again at higher speed to collect mitochondria. Depending on the planned experiments the resuspending buffer can be replaced by any other isotonic solution.
Animals were terminated in accordance with the U.K. "Animals (Scientific Procedures) Act." by cervical dislocation followed by decapitation. It is necessary to extract the heart immediately after decapitation of the animal, so that there is no delay between animal termination and heart extraction. If parallel isolation of liver mitochondria is expected the rats should be fasted overnight prior to the experiment.
<ol>
 <li> Prepare three beakers containing 40 ml of Washing Buffer. Cool them in the ice-salt bath for 3 to 5 min before proceeding to the next step. Make sure not to overfreeze them and use immediately just after clouds of ice crystals start to appear.</li>
 <li> Open the thorax of the decapitated rat and extract the heart. Make small incisions and then immediately transfer the heart to the ice-cold solution in the first beaker. Squeeze the heart to remove blood and put it into the second beaker. Repeat this operation for each heart.</li>
 <li> Dry the hearts on the filter paper. Remove all fat, clotted blood, auricles and fasciae and pool the ventricular tissue.</li>
 <li> Mince the pooled tissue with small curved scissors on the Petri dish on ice for around 5 min until the size of the particles is about 1-2 mm.</li>
 <li> Put the minced tissue into the tissue press and pass it through. Be aware that a substantial amount of the material can remain in the press so collect all the heart tissue from the walls and bottom of the press.</li>
 <li> Transfer the material into the beaker with 50 ml Washing Buffer and stir. Then filter the suspension through a nylon filter. Wash the minced tissue on the filter 3 times with the Washing Buffer and discard the filtrate.</li>
 <li> Transfer the washed tissue into 20 ml of Washing Buffer and place it in the ice bath on the magnetic stirrer. Add 0.5 ml of trypsin solution under constant stirring and start the timer. Prepare another magnetic stirrer, ice bath and a second 50 ml beaker.</li>
 <li> On the 4th minute briefly homogenise the suspension portion by portion using a small loosely fitted glass-Teflon homogeniser (10-15ml) with several up and down strokes to disperse the suspension. After homogenisation transfer the suspension into the second beaker. Repeat the procedure on the 9th minute, so that you perform the homogenisation of the suspension two times in total. After 15 min of incubation, add 10 ml of isolating buffer containing trypsin inhibitor and incubate for 1 min.</li>
 <li> Filter the suspension again through a nylon filter and save the filtrate. Collect the particulate fraction left on a filter and homogenise for 1 min in 15-20 ml of Washing Buffer.</li>
 <li> Combine the homogenate with the filtrate and centrifuge for 15 min at 600g.</li>
 <li> Carefully filter the resulting supernatant into a new centrifuge tube. Avoid contamination by the pellet and centrifuge again for 15 min at 8500g.</li>
 <li> After high speed centrifugation discard the supernatant and rinse the pellet 2-3 times with 1 ml of the buffer discarding the fluffy white outer rim layer. Break up the pellet with the spatula but avoid the red spot of erythrocytes at the bottom. If the blood cells got loos, remove the red bits from the suspension before homogenisation.</li>
 <li> Resuspend the pellet with a small homogeniser or alternatively by gentle pipetting. Dilute the suspension with more Isolationbuffer and centrifuge again for 15 min at 8500g.</li>
 <li> Discard the supernatant, resuspend the pellet in Resuspending Buffer (an isotonic buffer of choice) and centrifuge again.</li>
 <li> The resulting brown pellet contains washed intact mitochondria. Resuspend the pellet in a very small volume of isotonic buffer of your choice.</li>
</ol>
<img src="/files/ftp_upload/2202/2202fig1.jpg" alt="Figure 1" /> 
 Figure 1. Scheme demonstrating the step by step procedure of isolation of rat heart mitochondria. Top part, Stages 1-9: tissue disruption, trypsin treatment and homogenisation; bottom part, stages 10-15: differential centrifugation.

<ol>
	<li>Jacobus, W. E., Saks, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+kinase+of+heart+mitochondria:+changes+in+its+kinetic+properties+induced+by+coupling+to+oxidative+phosphorylation.">Creatine kinase of heart mitochondria: changes in its kinetic properties induced by coupling to oxidative phosphorylation.</a> Arch. Biochem. Biophys. 219, 167-178 (1982).</li><li>Gostimskaya, I. S., Grivennikova, V. G., Zharova, T. V., Bakeeva, L. E., Vinogradov, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+assay+of+the+intramitochondrial+enzymes:+use+of+alamethicin+for+permeabilization+of+mitochondria.">In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria.</a> Anal. Biochem. 313, 46-52 (2003).</li></ol>

No conflicts of interest declared.

Detailed rapid protocol for isolation of intact mitochondria with a high degree of coupling is described. Obtained preparation can be used for functional or structural research on cellular bioenergetics, proteomic studies or analysis of mitochondrial DNA and lipid content. Biophysical studies of active transport of ions and metabolic intermediates can also be performed. It is possible to further process the mitochondrial preparation in order to isolate submitochondrial particles, outer membrane or purify separate components of oxidative phosphorylation. The described method is a modification of the standard protocol of isolation by Jacobus and Saks1. The expected yield of the prepared mitochondria is approximately 10-15 mg of mitochondrial protein per heart and the respiratory control ratio on malate/glutamate of such preparation varies between 8 to 12 with State IV respiration of around 0.12 &mu;mol O2xmin-1xmg protein-1 at 23&deg;C in the medium comprising 0.25 M sucrose, 10 mM KCl, 0.2 mM EDTA, 5 mM potassium phosphate (pH 8.0) with 150 &mu;M ADP for initiation State III respiration (for the exact experimental conditions and additions, see ref. 2).
Special attention should be given to several technical details before the starting of the isolation procedure. It is crucial to use clean polycarbonate or polypropylene centrifuge tubes, therefore tubes should be thoroughly washed with a clean brush and hot water, however the detergent treatment should be kept at the minimum. Also, it is better to collect all necessary glassware and instruments in the cold room a day before the isolation.
During the isolation when transferring buffers from flasks to beakers, &#1072; special attention should be given to prevent drops of ice water into the solutions. When handling tissues it should be stressed that heart extraction should be carried out as soon as possible since even short-time delay between decapitation of the animal and cooling the tissue would result in partially uncoupled mitochondria. Rapid cooling and thorough washing of removed hearts is essential to obtain standard preparation, free from haemoglobin and erythrocyte NADase.
Trypsin is used for connective proteins degradation to increase susceptibility of the tissue to shearing force during homogenisation. Prolonged exposure of the tissue to protease should be avoided since it results in mitochondria of inferior quality.
After high speed centrifugations the walls of the centrifuge tubes should be wiped with tissue paper to remove any deposits of fat material accumulated on the walls.
For the final resuspension it is better to use a low volume of the final buffer in order to minimise loss of mitochondrial functions during storage (150 to 200 &mu;l of the buffer per heart). For studies of the transport of divalent cations no chelating agents must be present in the final preparation thus mitochondria should be washed for several times with EGTA-free medium and used within first hours after isolation.

preparation of highly coupled rat heart mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

Watch this Scientific Journal Video about Preparation of Highly Coupled Rat Heart Mitochondria at JoVE.com

Preparation of Highly Coupled Rat Heart Mitochondria

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

preparation-of-highly-coupled-rat-heart-mitochondria

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Biology

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

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+ homeostasis2, 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.

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.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...