We thank to Fabiana Benatti for her kind cooperation in the video. We also thank FAPESP (#2007/51228-0) and CNPq (#300133/2008-1) for the support to our researches on this area.

Introduction
The energy necessary for sustaining a physical effort comes from two metabolic sources: aerobic and anaerobic metabolism. While the aerobic metabolism is more efficient than the anaerobic metabolism (i.e., it produces a higher amount of ATP per mol of substrate), producing energy through anaerobic metabolism can provide a high amount of energy in a very short time period. This may be decisive for any situation that requires extremely fast movements. 
Each sport has specific characteristics in terms of motor skills that confer unique physiologic and metabolic demands for that particular sport. The most important aspect of the metabolic demand is the relative contribution of the energy systems to the total energy required for the activity. To determine the specific demand of each sport is crucial for developing optimized training models, nutritional strategies and ergogenic aids that may maximize athletic performance.
Some sports are relatively easy to be reproduced in a laboratory setting, thus it is possible to create a controlled environment in which athletes can be evaluated. This is the case of running and cycling, for example. Predictable movements compose these sports and, therefore, they are easy to be studied. Using some simple equipment, it is possible to mimic quite exactly the same movements that athletes perform in real situations, such as training and competitions. Indeed, these sports have been more extensively studied by exercise scientists and benefited with a more complete and reliable scientific literature. 
On the other hand, a number of sports are much more difficult to be reproduced in laboratory. These sports are unpredictable and dependent on the actions of the partner(s) and opponent(s). This leads to an inability to accurately reproduce the competitive conditions in the lab and an inability to assess these athletes in field during either training or competition. Maybe because of these problems, they have received much less attention from the scientists. This is the case of the majority of team sports and many individual sports1.
Considering these aspects, we aimed to describe how to assess the differential contribution of the energy systems in sports that are difficult to reproduce in controlled laboratory conditions. Because judo is a very complex and unpredictable sport, we will use judo as an example. However, the concepts shown here can be adapted to a number of different sports.
1. Physiological Measurements at Rest
<ol>
 <li>Measure the athlete&#x2019;s body mass before he/she initiates exercising.</li>
 <li>Before initiating the exercise, collect a small resting blood sample from earlobe or fingertip and keep it on ice until the whole experimental procedure is finished.</li>
 <li>Following, place the calibrated portable gas analyser at the most convenient position, which depends on the movements that the athlete will perform, and record resting or baseline oxygen consumption for five minutes. During the baseline measurement, the athlete has to stay quiet standing on his/her feet (if the exercise will be performed in a standing position) or sat in the equipment that will be used (if the exercise will be performed in a cycloergometer or in any similar equipment).</li>
</ol>
2. Physiological Measurements during Exercise
<ol>
 <li>After collecting resting blood sample and resting oxygen consumption, you may ask the athlete to start the specific exercise that you are studying. The portable gas analyser has to be placed in a position that will no interfere with exercise and that exercise will not damage the equipment. Continue measuring oxygen consumption throughout the exercise period.</li>
</ol>
3. Physiological Measurements after Exercise
<ol>
 <li>After collecting exercise oxygen consumption data, keep recording oxygen consumption for ten minutes before shutting the equipment down. Always recalibrate the gas analyser if more than one athlete is being evaluated in the same day.</li>
 <li>In order to identify the peak plasma lactate after exercise, collect small blood samples immediately after exercise, three, five and seven minutes after exercise. Keep them on ice until analysis.</li>
</ol>
4. Blood Samples Processing and Peak Plasma Lactate Determination
<ol>
 <li>All blood samples must be placed in microtubes containing a similar volume of a 2% NaF solution (i.e., if you are collecting 25 &mu;L of blood, place it in 25 &mu;L of 2% NaF).</li>
 <li>When data collection is finished, separate plasma from erythrocytes by spinning the samples for 5 minutes at 2000 g at 4&deg;C.</li>
 <li>Plasma lactate can be determined through a variety of methods2,3. In our lab, we use the electrochemical method with the aid of an automatized lactate analyser (Yellow Springs 1500 Sport, Ohio).</li>
</ol>
5. Calculations
<ol>
 <li>Calculate the net energy generated by the aerobic metabolism by subtracting rest oxygen consumption from exercise oxygen consumption. Oxygen consumption at rest is obtained by multiplying the average of the last 30 seconds of baseline oxygen consumption by the total exercise duration time. Then, calculate the area under the curve of exercise oxygen consumption by using the trapezoidal method. Finally, subtract the resting oxygen consumption from exercise oxygen consumption.</li>
 <li>The contribution of the anaerobic alactic metabolism (i.e., the ATP-CP pathway) can be considered as the fast component of excess post-exercise oxygen consumption4-6, as illustrated in Figure 1. Calculate the energy produced by the alactic system by fitting the kinetics of post-exercise oxygen consumption to a bi- or a monoexponential curve. This can be done with the aid of mathematics' software (e.g. Microcal Origin version 7.0). Choose by the mono- or bi-exponential curve based on the model that best fits to your data set (i.e., the lowest residue). Then, use the terms provided by the fitted equation (Equation 1) to calculate alactic contribution according to Equation 2. 
 <img src="/files/ftp_upload/3413/3413fig1.jpg" alt="Fiugre 1" /> 
 Figure 1. Schematic illustration of a typical oxygen consumption curve obtained at rest, during, and after exercise. 
 Equation 1: 
 <img src="/files/ftp_upload/3413/3413eq1.jpg" alt="Equation 1" /> 
 Equation 2: 
 <img src="/files/ftp_upload/3413/3413eq2.jpg" alt="Equation 2" /> 
 where VO2(t) is oxygen uptake at time t, VO2baseline is oxygen uptake at baseline, A is the amplitude, &delta; is the time delay, &tau; is a time constant, and 1 and 2 denote the fast and slow components, respectively.</li>
 <li>To calculate the contribution of the lactic anaerobic system, it is assumed that 1 mM of lactate above the resting values corresponds to 3 mL of oxygen consumed per kilogram of body mass7. Thus, calculate delta peak plasma lactate (i.e., peak plasma lactate minus resting plasma lactate) and multiply it by 3 and by the athlete&#x2019;s body mass. The obtained value of oxygen in mL is then converted to L and to energy (kJ), assuming that each 1 L of O2 is equal to 20.92 kJ.</li>
 <li>Finally, the result obtained by each energy system is summed so you have the total energy expenditure during the activity and the relative contribution of each of system can be calculated.</li>
</ol>
6. Representative Results
Figure 2 depicts a representative curve of oxygen consumption at rest, during exercise and after exercise. In the example used here, athletes performed three different judo techniques (o-uchi-gari, harai-goshi and seoi-nage) for five minutes (one throw every 15 s)8. This is a typical response to intermittent exercise. After the calculations, we obtained the final results on the contribution of the energy systems during judo exercises (Table 1).
Additional representative results are displayed in Table 2. In this example, indoor rock climbers of different competitive levels (i.e., recreational vs. elite) were assessed during a low-difficulty climb route. Individual results for one elite athlete and one recreational athlete are shown (Table 2).
<table border="1">
 <tbody>
 <tr>
 <td rowspan="2">&nbsp;</td>
 <td colspan="2">Seoi-nague</td>
 <td colspan="2">Harai-goshi</td>
 <td colspan="2">O-uchi-gari</td>
 </tr>
 <tr>
 <td>kJ</td>
 <td>%</td>
 <td>kJ</td>
 <td>%</td>
 <td>kJ</td>
 <td>%</td>
 </tr>
 <tr>
 <td>Anaerobic alactic</td>
 <td>46 &plusmn; 20</td>
 <td>16.3 &plusmn; 2.8</td>
 <td>43 &plusmn; 21</td>
 <td>16.1 &plusmn; 2.7</td>
 <td>36 &plusmn; 22</td>
 <td>14.6 &plusmn; 2.8</td>
 </tr>
 <tr>
 <td>Aerobic</td>
 <td>223 &plusmn; 66</td>
 <td>82.2 &plusmn; 2.9</td>
 <td>211 &plusmn; 66</td>
 <td>82.3 &plusmn; 3.8</td>
 <td>196 &plusmn; 74</td>
 <td>84.0 &plusmn; 3.8</td>
 </tr>
 <tr>
 <td>Anaerobic lactic</td>
 <td>4 &plusmn; 2</td>
 <td>1.5 &plusmn; 0.7</td>
 <td>5 &plusmn; 5</td>
 <td>1.6 &plusmn; 1.4</td>
 <td>4 &plusmn; 4</td>
 <td>1.5 &plusmn; 1.1</td>
 </tr>
 <tr>
 <td>Total</td>
 <td>273 &plusmn; 86</td>
 <td> - </td>
 <td>259 &plusmn; 91</td>
 <td> - </td>
 <td>237 &plusmn; 99</td>
 <td> - </td>
 </tr>
 <tr>
 <td>Total (kJ/min)</td>
 <td>51.9 &plusmn; 8.7</td>
 <td> - </td>
 <td>49.4 &plusmn; 8.9</td>
 <td> - </td>
 <td>45.3 &plusmn; 19.6</td>
 <td> - </td>
 </tr>
 </tbody>
</table>
Table 1. Representative results of total energy expenditure and the contribution of the energy systems during three different judo exercises.
<table border="1">
 <tbody>
 <tr>
 <td>Competitive Level</td>
 <td>Aerobic (%)</td>
 <td>Anaerobic Lactic (%)</td>
 <td>Anaerobic Alactic (%)</td>
 <td>Total (kJ)</td>
 <td>Total (kJ/s)</td>
 </tr>
 <tr>
 <td>Elite</td>
 <td>40</td>
 <td>8</td>
 <td>52</td>
 <td>70.4</td>
 <td>1.00</td>
 </tr>
 <tr>
 <td>Recreational</td>
 <td>40</td>
 <td>15</td>
 <td>45</td>
 <td>96.1</td>
 <td>1.15</td>
 </tr>
 </tbody>
</table>
Table 2. Representative individual data of total energy expenditure and the contribution of the energy systems during a low-difficulty climb route.
<img src="/files/ftp_upload/3413/3413fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative results obtained during a 5-minute judo exercise.

<ol>
	<li>Franchini, E., Del Vecchio, F. B., Matsushigue, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+profiles+of+elite+judo+athletes.">Physiological profiles of elite judo athletes.</a> Sports Med. 41, 147-166 (2011).</li><li>Bergmeyer, H. U., Bergmeyer, J., Grassl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of enzymatic analysis. , (1983).</li><li>Passonneau, J. V., Lowry, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Analysis.">Enzymatic Analysis.</a> A Practical Guide. , (1993).</li><li>Beneke, R., Pollmann, C., Bleif, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+anaerobic+is+the+Wingate+Anaerobic+Test+for+humans.">How anaerobic is the Wingate Anaerobic Test for humans.</a> Eur. J. Appl. Physiol. 87, 388-392 (2002).</li><li>Beneke, R., Beyer, T., Jachner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+karate+kumite.">Energetics of karate kumite.</a> Eur. J. Appl. Physiol. 92, 518-523 (2004).</li><li>Bertuzzi, R. C. D., Franchini, E., Kokubun, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+system+contributions+in+indoor+rock+climbing.">Energy system contributions in indoor rock climbing.</a> Eur. J. Appl. Physiol. 101, 293-300 (2007).</li><li>di Prampero, P. E., Ferretti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+energetics+of+anaerobic+muscle+metabolism:+a+reappraisal+of+older+and+recent+concepts.">The energetics of anaerobic muscle metabolism: a reappraisal of older and recent concepts.</a> Respir. Physiol. 118, 103-115 (1999).</li><li>Franchini, E., Bertuzzi, R. C. D., Degaki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+Expenditure+in+Different+Judo+Throwing+Techniques.+Proceedings+of+first+joint+international+pre-Olympic+conference+of+sports+science+and+sports+engineering,+vol+II.">Energy Expenditure in Different Judo Throwing Techniques. Proceedings of first joint international pre-Olympic conference of sports science and sports engineering, vol II.</a> Bio-mechanics and sports engineering. , 55-60 (2008).</li><li>Calmet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+ecological+research+in+judo.">Developing ecological research in judo.</a> Percept. Mot. Skills. 105, 646-648 (2007).</li><li>Mello, F. D., Bertuzzi, R. C., Grangeiro, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+systems+contributions+in+2,000+m+race+simulation:+a+comparison+among+rowing+ergometers+and+water.">Energy systems contributions in 2,000 m race simulation: a comparison among rowing ergometers and water.</a> Eur. J. Appl. Physiol. 105, 615-619 (2009).</li><li>Bertuzzi, R. C., Franchini, E., Ugrinowitsch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+MAOD+using+only+a+supramaximal+exhaustive+test.">Predicting MAOD using only a supramaximal exhaustive test.</a> Int. J. Sports Med. 31, 477-481 (2010).</li></ol>

The authors declare they have no conflict of interest regarding this study.

The method we have shown hare can be used for both continuous and intermittent exercise. The great advantage of the method is that it can be adapted to exercises and sports that are difficult to be mimicked in controlled laboratory settings. Furthermore, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study9. For example, a recent study by Mello et al.10 showed that the glycolytic contribution in a 2000 m on water rowing race is of only 7%, which means that rowing performance is mainly dependent on the aerobic metabolism. Similarly, a study by Beneke et al.4 confirmed that the main source of energy during one of the most used anaerobic tests, the Wingate Anaerobic Test, is the anaerobic metabolism (20% aerobic; 30% alactic and 50% glycolytic). Recent studies by our group have also characterized the energy contributions of indoor climbing6 and judo8, as reported in this example. Indeed, the knowledge on the energetic contribution is critical for the development of ergogenic strategies, training organization or even for validating a test.
This method has some limitations. First, the cost of the equipment is somewhat high, and specialized trained personnel are required. Second, although most sports can be mimicked with this technique, it is not any type of exercise that can be studied using the portable gas analyzer. Finally, as plasma lactate does not exactly represent the total lactate produced by skeletal muscle during activity, the results obtained by this procedure can be considered as an estimative of the metabolic demand during exercise, rather than a precise quantification of the energetic contribution. Nonetheless, this is the only validated method available11 capable of distinguishing the contribution of the three different energy systems.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>YSI 1500 Sport</td>
			<td>Yellow Springs</td>
			<td>This equipment allows a quick and easy plasma lactate determination</td>
		</tr>
		<tr>
			<td>K4 b2</td>
			<td>Cosmed</td>
			<td>This equipment is essential for measuring oxygen consumption throughout the exercise</td>
		</tr>
		<tr>
			<td>Software Microcal 6.0</td>
			<td>Origin</td>
			<td>This software (or any other with similar capabilities) will be useful for the calculations</td>
		</tr>
	</tbody>
</table>

determining the contribution of the energy systems during exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

Watch this Scientific Journal Video about Determining the Contribution of the Energy Systems During Exercise at JoVE.com

Determining the Contribution of the Energy Systems During Exercise

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

determining-the-contribution-of-the-energy-systems-during-exercise

Research

JoVE Journal

Biology

41.1K Views.  University of Sao Paulo. This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

Video: Determining the Contribution of the Energy Systems During Exercise

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...