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.
1. Cells Injection
The cells are metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell. One group will serve as the control, with running media added as control "compounds".
<ol>
<li>The first injection is oligomycin. Oligomycin inhibits ATP synthesis by blocking the proton channel of the Fo portion ATP synthase (Complex V). In mitochondrial research, it is used to prevent state 3 (phosphorylating) respiration. With cells, it can be used to distinguish the percentage of O2 consumption devoted to ATP synthesis and the percentage of O2 consumption needed to overcome the natural proton leak across the inner mitochondrial membrane.</li>
<li>The second injection is FCCP. FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) is an ionophore that is a mobile ion carrier. It is an uncoupling agent because it disrupts ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of the proton channel of ATP synthase (Complex V). This collapse of the mitochondrial membrane potential leads to a rapid consumption of energy and oxygen without the generation of ATP. In this case, both OCR and ECAR will increase, OCR due to uncoupling, and ECAR as the cells attempt to maintain their energy balance by using glycolysis to generate ATP. 
FCCP treatment can be used to calculate the "spare" respiratory capacity of cells that is defined as the quantitative difference between maximal uncontrolled OCR and the initial basal OCR. It has been proposed that the maintenance of some spare respiratory capacity even under conditions of maximal physiological or pathophysiological stimulus is a major factor defining the vitality and/or survival of cells. The ability of cells to respond to stress under conditions of increased energy demand is in a large part influenced by the bioenergetic capacity of mitochondria. This bioenergetic capacity is deter-mined by several factors, including the ability of the cell to deliver substrate to mitochondria and the functional capacity of enzymes involved in electron transport</li>
<li>In the third injection, rotenone, a Complex I inhibitor, is added to the cells. This will shut down mitochondrial respiration and enable both the mitochondrial and non-mitochondrial fractions contributing to respiration to be calculated. One will observe a decrease in OCR due to impaired mitochondrial function, with a concomitant increase in ECAR as the cell shifts to a more glycolytic state in order to maintain its energy balance 
Rotenone is a mitochondrial inhibitor that prevents the transfer of electrons from the Fe-S center in Complex I to ubiquinone (Coenzyme Q). This inhibition of Complex I prevents the potential energy in NADH from being converted to usable energy in the form of ATP. 
</li>
</ol>
2. Reagents and Materials
<ol>
<li>Oligomycin, FCCP, and Rotenone Solutions (Seahorse Mito Stress Test Kit)</li>
<li>DMEM Running Media (Seahorse #100965-000)</li>
<li>DMSO (Sigma D8418)</li>
<li>Distilled Water (Gibco 15230-170)</li>
<li>Calibration buffer (Seahorse Bioscience)</li>
</ol>
3. Growth Medium
<ol>
<li>500 mL DMEM (Gibco 11965-092)</li>
<li>10% FBS (Hyclone SH90070.03)</li>
<li>5 mL Penn/Strep (Gibco 15140-122)</li>
<li>5 mL Sodium Pyruvate (Sigma S8636)</li>
<li>5 mL Glutamax (Gibco 35050-061)</li>
</ol> 
4. Seeding Protocol
<ol>
<li>Cells are seeded in XF96 cell cultures plates with 10,000 cells/well in 100 &mu;L of growth medium and placed in 37&deg;C incubator with 10% CO2.</li>
<li>Cells will adhere to the XF96 cell culture plate within 1 hour.</li>
<li>Assay cells in XF96 24 hours after seeding.</li>
</ol>
5. Preparation of Assay Template
<ol>
<li>Using the Assay Wizard (Appendix I), generate a template with the following group layout: 
<img src="/files/ftp_upload/2511/2511fig1.jpg" alt="Figure 1" /> 
Figure 1. Well grid layout identifying column and group assignments</li>
</ol>
6. Compound Preparation
<ol>
<li>Prepare the following compounds in XF DMEM Assay media as follows: 
10 uM Oligomycin, 30.0 uM FCCP, 20.0 &mu;M Rotenone, 
These concentrations represent the 10X dilution that will be made when the compounds are injected into the well. The working concentrations are: 
1 uM Oligomycin, 3.0 uM FCCP, 2.0 &mu;M Rotenone
</li>
</ol>
7. Media Change and Cell Preparation
<ol>
<li>Place the cell plate on the XF Prep Station</li>
<li>Set the final volume of medium to 160 &mu;L per well.</li>
<li>Incubate in 37&deg;C incubator without CO2 for 60 minutes to allow 	cells to pre-equilibrate with the assay medium.</li>
</ol>
8. Loading Sensor Cartridge
<ol>
<li>Warm compounds to 37&deg;C prior to loading sensor cartridge and 	load the compounds into the injector ports as follows:
<ol>
<li>Columns 1-4: Load 16, 18, and 20 &mu;L of XF Assay media (DMEM) into ports A, B and C, respectively.</li>
<li>Columns 5-12: 
Load 16 &mu;L of Oligomycin into ports A 
Load 18 &mu;L of FCCP into ports B 
Load 20 &mu;L of Rotenone into ports C</li>
</ol></li>
</ol>
9. Protocol Commands
<table border="1">
<tbody>
 <tr>
 <td>Command</td>
 <td>Time (min)</td>
 <td>Port</td>
 </tr>
 <tr>
 <td>Calibrate</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Equilibrate</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Loop Start</td>
 <td>3X</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mix</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wait</td>
 <td>2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Measure</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Loop End</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inject</td>
 <td>&nbsp;</td>
 <td>A</td>
 </tr>
 <tr>
 <td>Loop Start</td>
 <td>2X</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mix</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wait</td>
 <td>2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Measure</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inject</td>
 <td>&nbsp;</td>
 <td>B</td>
 </tr>
 <tr>
 <td>Loop Start</td>
 <td>2X</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mix</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wait</td>
 <td>2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Measure</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inject</td>
 <td>&nbsp;</td>
 <td>C</td>
 </tr>
 <tr>
 <td>Loop Start</td>
 <td>2X</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mix</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wait</td>
 <td>2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Measure</td>
 <td>3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>End</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>
Table 1. Protocol commands

<ol>
	<li>Choi, W. S., Gerencser, A. A., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+isolated+cerebrocortical+nerve+terminals+on+a+microgram+scale:+spare+respiratory+capacity+and+stochastic+mitochondrial+failure.">Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure.</a> J Neurochem. 109, 1179-1191 (2009).</li><li>Hill, B. G., Dranka, B. P., Zou, L., Chatham, J. C., Darley-Usmar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+bioenergetic+reserve+capacity+in+response+to+cardiomyocyte+stress+induced+by+4-hydroxynonenal.">Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal.</a> Biochem J. 424, 99-107 (2009).</li><li>Liu, J., Cao, L., Chen, J., Song, S., Lee, I. H., Quijano, C., Liu, H., Keyvanfar, K., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+regulates+mitochondrial+function+and+the+DNA+damage+response+pathway.">Bmi1 regulates mitochondrial function and the DNA damage response pathway.</a> Nature. , 459-7245 (2009).</li><li>Malmgren, S., Nicholls, D. G., Taneera, J., Bacos, K., Koeck, T., Tamaddon, A., Wibom, R., Groop, L., Ling, C., Mulder, H., Sharoyko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+coupling+between+glucose+and+mitochondrial+metabolism+in+clonal+beta-cells+is+required+for+robust+insulin+secretion.">Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion.</a> J Biol Chem. 284, 32395-32404 (2009).</li><li>Dranka, B. P., Hill, B. G., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reserve+capacity+in+endothelial+cells:+The+impact+of+nitric+oxide+and+reactive+oxygen+species.">Mitochondrial reserve capacity in endothelial cells: The impact of nitric oxide and reactive oxygen species.</a> Free Radic Biol Med. 48, 905-914 (2010).</li><li>Perez, J., Hill, B. G., Benavides, G. A., Dranka, B. P., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cellular+bioenergetics+in+smooth+muscle+cell+proliferation+induced+by+platelet-derived+growth+factor.">Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor.</a> Biochem J. 428, 255-267 (2010).</li><li>Mor&#225;n, M., Rivera, H., S&#225;nchez-Arag&#243;, M., Bl&#225;zquez, A., Merinero, B., Ugalde, C., Arenas, J., Cuezva, J. M., Mart&#237;n, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+bioenergetics+and+dynamics+interplay+in+complex+I-deficient+fibroblasts.">Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts.</a> Biochim Biophys Acta. , 1802-185 (2010).</li><li>C&#225;rdenas, C., Miller, R. A., Smith, I., Bui, T., Molg&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+regulation+of+cell+bioenergetics+by+constitutive+InsP3+receptor+Ca2++transfer+to+mitochondria.">Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.</a> Cell. , 142-142 (2010).</li></ol>

Min Wu, Per Bo Jensen, George W. Rogers, and David A. Ferrick are employees of Seahorse Bioscience, which produces reagents and instrumentation featured in this article.

This assay is derived from the classic experiment to probe mitochondrial function and serves as a framework with which to build more complex experiments aimed at understanding various changes in cell metabolism, mitochondrial function, and overall bioenergetics.
All compounds used in this experiment should be optimized for the concentration that provides the maximal effect. That is, one must perform separate titration experiments to ascertain these values. This is especially important with FCCP, as the titration curve tends to be quite sharp, and too much FCCP can actually diminish responses in OCR. Typical ranges (final concentrations) to test would be:
<ul>
<li>0.1	- 1.0 ug/ mL of Oligomycin</li>
<li>0.1	- 5.0 uM FCCP</li>
<li>0.1	- 1.0 uM Rotenone</li>
</ul>
Note that the responses to each compound above (especially FCCP) will be influenced by the assay media composition (base type, [glucose], [pyruvate], presence/absence of BSA, etc). Further, if the XF assay media composition is changed, optimization will need to be re-performed. The presence and concentration of pyruvate is especially important in obtaining the maximal respiratory capacity due to FCCP. Seahorse Bioscience has observed in a number of cells lines that omission of pyruvate abrogates the ability of cells to respond maximally (above baseline) to FCCP. Typically, concentrations of 1-10 mM pyruvate should be tested to understand the optimal concentration of pyruvate to obtain maximal respiration. Note that [pyruvate] AND [glucose] may need to be "cross-titrated" to obtain the optimal media conditions for the experiment.
Typical results of this experiment are presented below in a graph showing OCR vs. time and another showing ECAR vs. time:
<img src="/files/ftp_upload/2511/2511fig2.jpg" alt="Figure 2" /> 
Figure 2. OCR vs. Time
<img src="/files/ftp_upload/2511/2511fig3.jpg" alt="Figure 3" /> 
Figure 3. ECAR vs. Time
Here we observed the expected responses in OCR and ECAR as the cells are treated with each successive compound. For oligomycin, OCR decreases as a result of blocking ATP synthesis at mitochondrial Complex V. Since the cells are unable to synthesize ATP via OXPHOS, they revert to glycolysis to meet their demand for ATP, thus we observe an increase in ECAR. As shown previously, FCCP acts as an uncoupling agent. Since the cells must now overcome the proton leak across the inner mitochondrial membrane, OCR increases significantly as more O2 is consumed to pump the excess protons back across the mitochondrial membrane. Finally, rotenone inhibits mitochondrial Complex I and Complex III, respectively, which causes the flow of electrons to cease in the electron transport chain, and thus the consumption of O2 is drastically reduced.
<img src="/files/ftp_upload/2511/2511fig4.jpg" alt="Figure 4" /> 
Figure 4. Respiration parameters
Beyond the expected changes in respiration and ECAR, a number of respiratory parameters may be obtained from this data. This is summarized in the figure above:
Here we see that we may obtain information about the basal respiration of the cells, the percent of O2 consumption devoted to ATP production as well as the amount devoted to maintaining the proton gradient (due to H+ leak). Further, we may obtain the maximal respiratory rate under conditions of uncoupled respiration (sometimes referred to as spare respiratory capacity) and finally, we can determine the amount of O2 consumption not due to mitochondrial processes.
A rapidly growing number of studies are employing this mitochondrial profile to assess cellular bioenergetics, identify mitochondrial dysfunction and to predict the ability of cells to respond to stress and/or insults. For more information and details about this experimental method and the idea of spare respiratory capacity, please see refer to the following publications 1-8.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Oligomycin, FCCP, Rotenone and Antimycin A Solutions</td>
<td>&nbsp;</td>
<td>Seahorse Bioscience</td>
<td>&nbsp;</td>
<td>Seahorse Mito Stress Test Kit</td>
</tr>
<tr>
<td>DMEM Running Media</td>
<td>&nbsp;</td>
<td>Seahorse Bioscience</td>
<td>100965-000</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D8418</td>
<td>&nbsp;</td>
<tr>
<td>Distilled Water</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15230-170</td>
<td>&nbsp;</td>
<tr>
<td>Calibration buffer</td>
<td>&nbsp;</td>
<td>Seahorse Bioscience</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

bioenergetic profile experiment using c2c12 myoblast cells

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.

Watch this Scientific Journal Video about Bioenergetic Profile Experiment using C2C12 Myoblast Cells at JoVE.com

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

bioenergetic-profile-experiment-using-c2c12-myoblast-cells

Research

JoVE Journal

Biology

50.3K visualizações.  Novato, CA. A descrição de um método para caracterização da função mitocondrial em células é fornecido. O perfil mitocondrial gerado fornece quatro parâmetros da função mitocondrial, que pode ser medido em um experimento: taxa de respiração basal, ATP-linked respiração, vazamento de prótons, e capacidade de reserva.

Vídeo: Bioenergetic Profile Experiment using C2C12 Myoblast Cells

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...