The authors would like to acknowledge Dr. Kevin Bittman for his guidance in establishing the Seahorse XFp in the lab. National Institutes of Health grant GM088213 supported this work.

NOTE: Figure 2 provides a schematic overview of the full protocol.
1. Growth and synchronization of nematode population9,10
<ol>
	<li>Transfer L4 larvae of desired genetic backgrounds (e.g., N2 [wild type] and sel-12 animals) onto nematode growth media (NGM) plates (see Table 1 for recipe) freshly seeded with a lawn of Escherichia coli (OP50)11. Use at least two 100 mm or three 60 mm plates for each strain. Incubate the worms at the appropriate growth temperature (between 15&#8211;25 &#176;C) for 3&#8211;4 days or until plates are concentrated with large number of eggs and gravid worms.</li>
	<li>Wash the eggs and worms off the plates using approximately 6 mL of M9 buffer (see Table 1 for recipe) per 100 mm plate of nematodes and transfer them with glass Pasteur pipettes into individual 15 mL centrifuge tubes for each strain. Spin these tubes down for 3 min at 6,180 x g and aspirate out the M9 buffer, retaining just the animals and eggs pellet.</li>
	<li>Add 3&#8211;4 mL of bleach solution (see Table 1 for recipe) to each tube and intermittently vortex for 6 min. Add M9 buffer to fill each tube and spin at 6,180 x g for 1 min and aspirate supernatant. Repeat this wash with M9 three times and move the egg pellet to a fresh 15 mL tube containing approximately 9 mL of M9 buffer.</li>
	<li>Synchronize the freshly hatched worms by nutating at 20 &#176;C for 16&#8211;48 h. Spin these tubes down at 6,180 x g for 1.5 min and put the synchronized L1 animals down on individual NGM plates freshly seeded with a lawn of OP50 (approximately 6,000&#8211;10,000 animals per 100 mm plate) and keep at 20 &#176;C.</li>
	<li>These animals will reach the L4 larval stage after ~42 h. At this time, move the L4 larvae using a platinum pick to OP50 seeded NGM plates containing 0.5 mg/mL 5-fluoro-2&#8217;-deoxyuridine (FUdR) to prevent them from producing progeny. 
	NOTE: Make sure that within an experiment all strains are synchronized for a similar amount of time. FUdR treatment sterilizes the animals and prevents egg laying and progeny production, which could influence OCR. These sterilized animals will be analyzed the next day (day 1 adult animals at ~66 h) for the assay. FUdR has been reported to impact physiology and lifespan of certain mutant worms. Therefore, this should be taken into consideration when the drug is used for sterilization12,13,14. Worms can also be sterilized by the means of feminizing mutations such as fem-1(hc17) or fem-3(e2006). However, these mutations might impact mitochondrial function.</li>
</ol>
2. Preparation&#160;of compounds to be injected and hydration of probes
NOTE: During the assay run, both basal and maximal respiration rates of the nematodes are measured. Maximal respiration is triggered in the animals upon the addition of carbonyl cyanide-4 (trifluormethoxy)phenylhydrazone (FCCP), an uncoupling ionophore that disturbs the mitochondrial membrane potential and thus ATP synthesis by transporting protons through the mitochondrial membrane, while allowing proton pumping, electron transport, and oxygen consumption to proceed4,15 uncoupled from ATP synthesis (Figure 1). The final step in the assay involves the addition of sodium azide (NaN3), a drug that inhibits complexes IV and V in the ETC, allowing one to determine non-mitochondrial respiration16 (Figure 1). The following steps can be performed the day before the actual assay run.
<ol>
	<li>Prepare 1 mL stock solutions of the FCCP (10 mM in dimethyl sulfoxide [DMSO]: 1000x the final assay concentration) and NaN3 (400 mM in dH2O: 10x final assay concentration) and store at -20 &#176;C. 
	NOTE: Run a concentration curve to optimize the concentration of FCCP required to elicit maximal OCR response for each instrument and laboratory setting.</li>
	<li>Hydrate the sensor cartridge by adding 200 &#181;L of dH2O to each well (and the surrounding reservoir) in the plate, ensuring that the sensor probes are submerged in the dH2O and store overnight at room temperature. 
	NOTE: The cartridge probes can be left submerged for up to 72 h but in the case of a prolonged hydration, the plates should be wrapped in paraffin film and stored at 4 &#176;C. If overnight hydration is not possible, the sensor cartridge should be hydrated for at least 4 h before assay.</li>
	<li>Turn off the heating element within the respirometer interface and store the instrument inside a 15 &#176;C incubator overnight to lower core temperature within the instrument to prevent the animals from overheating during the assay run. 
	NOTE: The respirometer is equipped with a heating element but cannot be cooled. Within a 15 &#176;C incubator, the respirometer will have a stable temperature between 18&#8211;22 &#176;C, which is a healthy temperature for C. elegans maintenance.</li>
</ol>
3. Drug loading and cartridge equilibration
<ol>
	<li>Pipette out and discard the dH2O used to hydrate the sensor probes and replace it with 200 &#181;L of the calibrant solution (pH 7.4) in each well. Dilute the FCCP stock solution to 100 &#181;M FCCP in dH2O and add 20 &#181;L of the diluted solution to the injection port A in the sensor cartridge. Add 22 &#181;L of 400 mM sodium azide to port B of the senor cartridge.</li>
	<li>Turn the respirometer on and on the home screen select Start; the templates page will appear. On the templates page, select Blank or a previously designed template; the Groups page will appear. On the Groups page, select the wells A and H as the background wells and assign the remaining 6 wells into appropriate groups according to the experimental plan.</li>
	<li>Push the arrow on the lower right corner to go to the Protocol page. On the Protocol page, ensure that the Equilibrate, Basal and Injections 1 and 2 buttons are selected. On this page, adjust the number of readings of basal OCR, as well as maximal OCR (after injection 1 with FCCP) and non-mitochondrial OCR (after injection 2 with sodium azide). 
	NOTE: Each measurement is preceded by a mix and wait step and the time frames for these parameters are shown in Table 2.</li>
	<li>Select the arrow on the lower right corner and a prompt to load the sensor cartridge plate will appear. Ensure that the sensor cartridge plate is loaded in the right orientation, following the instructions on the reminder prompt screen. The respirometer will now equilibrate the cartridge. 
	NOTE: This equilibration provides ample time to place the animals to be assayed into appropriate wells of the cell plate.</li>
</ol>
4. Preparation of worm plate and assay run
<ol>
	<li>Add 200 &#181;L of M9 buffer into each of the 8 wells of the cell plate and into the reservoirs surrounding the wells.</li>
	<li>Pick ~100 worms from each strain onto unseeded NGM plates and allow to rest for 2-3 min. Wet the end of the platinum pick with M9 buffer and pick 20 age-synchronized animals into wells B-G, leaving the background wells A and H empty. 
	NOTE: After loading each well, wait approximately 2 min to allow the animals to settle into the bottom of the wells. It is also advisable that a worm number curve be run to optimize the worm number per well for each instrument and laboratory setting.</li>
	<li>By now, the respirometer should be calibrated and by clicking OK on the screen, the plate containing calibration buffer is ejected and can be removed from the respirometer, while the sensor cartridge stays inside the instrument. Replace the calibrant plate with the plate containing animals. Load plate and close the door by hitting Continue and allow the assay to run.</li>
</ol>
5. Post-experiment data analysis
<ol>
	<li>Once the assay run is complete, follow the prompts on the screen and remove the cell plate and sensor cartridge and insert a flash drive into the USB port to save the run data in the .war format. Remove the sensor cartridge and allow the animals to settle to the bottom of the wells for approximately 2 min. Place the cell plates under a stereo dissecting microscope and count the number of animals per well.</li>
	<li>Open the analysis software on the computer and open the normalization tab to normalize OCR to animal number. Apply appropriate labels for the various groups under the modify tab and export the file as a Prism file for further analysis. 
	NOTE: The average of the first five measurements, before the addition of FCCP is the basal OCR, while the average of the five measurements after FCCP addition is the maximal OCR and the average of the last five measurements (minimum of two measurements should be done) after sodium azide addition is the non-mitochondrial respiration rate. The assay should be repeated three times to ensure reproducibility.</li>
</ol>

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The authors have nothing to disclose.

Mitochondrial respiration is an insightful indicator of mitochondrial function; therefore, being able to measure the oxygen consumption rates in a biological system, whether in vitro or in vivo is highly valuable. Respirometers sense oxygen levels using porphyrin-based phosphors that get quenched by oxygen or via amperometric oxygen sensors that rely on the generation of an electric current proportional to oxygen pressure. Clark electrode falls into the latter category and has been used extensively in literature, especially while analyzing respiration in C. elegans. However, the need for a large sample size and the inability to assess more than one sample at a time makes amperometric oxygen sensors inefficient.
This protocol provides a simple guide to measure OCR in live, intact C. elegans without isolating mitochondria, a process that could potentially impact the mitochondrial membrane potential and, therefore, the OCR. Given that animals exhibit different OCR through various life stages, animals used in this protocol should be age synchronized. Younger animals have lower OCR compared to adult animals and the OCR levels can drop again as animals age further3. C. elegans are also sterilized by the use of FUdR to ensure that OCR are not confounded by the presence of progeny. Nevertheless, if animals of varying sizes are to be examined, strategies for normalization need to be addressed.
This protocol utilizes a platinum pick to transfer animals to the assay plate. In contrast to liquid transfer of animals, this manipulation enables the researcher to carefully examine and determine the health of the animals before and during the transfer. Also, it allows for better control of worm number and prevents the introduction of eggs and carcasses. Since the animals are alive and active during the assay run, variability is likely to arise from probe positioning. Assays should therefore be done in triplicate and should be repeated a minimum of three times. Also, double checking animal count post analysis is important for normalizing to animal number. A major drawback of this protocol is the inability to compare more than two samples at once, without compromising the number of replicates within a single assay. Despite this limitation, this protocol can be a very powerful research tool in analyzing OCR when comparing two genotypes or conditions. Moreover, this protocol could be easily adapted to examine the OCR of animals grown on different food sources, supplements, or drug treatments.

Adenosine triphosphate (ATP), the main source of cellular energy, is produced in the mitochondria by enzymes in the electron transport chain (ETC) located in the inner mitochondrial membrane. Pyruvate, a key metabolite utilized for mitochondrial ATP production, is imported into the mitochondrial matrix where it is decarboxylated to produce acetyl coenzyme A (CoA). Subsequently, acetyl CoA enters the citric acid cycle resulting in the generation of nicotinamide adenine dinucleotide (NADH), a key electron carrier molecule. As electrons from NADH are passed to oxygen via the ETC, protons build up in the mitochondrial intermembrane space, which results in the generation of an electrochemical gradient across the membrane. These protons will then flow from the intermembrane space across this electrochemical gradient back into the mitochondrial matrix through the proton pore of the ATP synthase, driving its rotation and the synthesis of ATP1 (Figure 1).
Mitochondrial function is not limited to energy production but is also crucial for calcium homeostasis, reactive oxygen species (ROS) scavenging, and apoptosis, critically positioning their function in organismal health2. Mitochondrial function can be assessed using a variety of assays, including but not limited to analyses that measure mitochondrial membrane potential, ATP and ROS levels, and mitochondrial calcium concentrations. However, these assays provide a single snapshot of mitochondrial function and therefore might not provide a comprehensive view of mitochondrial health. Since oxygen consumption during ATP generation is reliant on a myriad of sequential reactions, it serves as a superior indicator of mitochondrial function. Interestingly, variations in oxygen consumption rates have been observed as a result of mitochondrial dysfunction3,4,5.
Oxygen consumption rates (OCR) of living samples can be measured using techniques that can be broadly divided into two groups: amperometric oxygen sensors and porphyrin-based phosphors that can be quenched by oxygen6. Amperometric oxygen sensors have been used extensively to measure OCR in cultured cells, tissues, and in model systems, such as C. elegans. However, porphyrin-based phosphors containing respirometers possess the following advantages: (1) they allow for a side by side comparison of two samples in triplicate, (2) they require smaller sample size (e.g., 20 worms per well versus ~2,000&#8722;5,000 worms in the chamber)7, and (3) the respirometer can be programmed to do four different compound injections at desired times throughout the experimental run, eliminating the need for manual application.
In this protocol, steps involved in using a porphyrin-based oxygen-sensing respirometer to measure OCR in live, intact C. elegans are described. While there is a written protocol for the use of the large format, high throughput respirometer8, this protocol has been adapted for use with a more budget friendly, accessible, and smaller scale instrument. This protocol is particularly useful for assessing the difference in OCR between two strains, where high-throughput screening is not required and its use would be excessive.

<table>
	<tbody>
		<tr>
			<td>100 mm, 60 mm Petri dishes</td>
			<td>Kord-Valmark Labware Products</td>
			<td>2900, 2901</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>Globe Scientific</td>
			<td>6285</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>22 &#215; 22 mm coverslip</td>
			<td>Globe Scientific</td>
			<td>1404-10</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD, Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD, Bacto</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto yeast extract</td>
			<td>BD, Bacto</td>
			<td>212705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2&#183;2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)</td>
			<td>Abcam</td>
			<td>ab120081</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>C314-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water (dH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Thomas Scientific</td>
			<td>C987Y85</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>Krackeler Scientific</td>
			<td>6-72050-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4&#183;7H2O)</td>
			<td>Fisher Scientific</td>
			<td>BP213-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic (K2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>BP359-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous (Na2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP332-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFp Analyzer</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFp FluxPak</td>
			<td>Agilent</td>
			<td>103022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of oxygen consumption rates in intact caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Using the protocol described herein, OCR of wild type animals and three different sel-12 mutant strains were determined. sel-12 encodes the C. elegans ortholog of presenilin17. Mutations in human presenilin are the most common genetic aberration associated with the development of familial Alzheimer&#39;s disease18. Our studies have shown elevated mitochondrial calcium levels in sel-12 mutant animals compared to wild type animals3. Since calcium dysregulation can result in altered mitochondrial function3,19,20, OCR in sel-12 mutant and wild type animals were measured to examine the effect of sel-12 mutations on mitochondrial function and health. Wild type animals consistently showed basal OCR rates below 5 pmol/min/worm, while all three strains of sel-12 mutants showed significantly elevated OCR of ~7 pmol/min/worm (Figure 3 and Figure 4). Upon the addition of FCCP, as expected, there was an increase in the OCR of wild type as well as sel-12 mutants (Figure 3 and Figure 5). Wild type animals showed maximum OCR of ~7 pmol/min/worm, while sel-12 mutants had OCR of ~10 pmol/min/worm (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59277/59277fig1.jpg" /> 
Figure 1: Schematic of the major players involved in cellular respiration and the effect of FCCP and sodium azide. Transfer of electrons from NADH to the ETC complex I results in the generation of an electrochemical gradient across the inner mitochondrial membrane as protons get pumped across it. Protons flowing back into the mitochondrial matrix from the intermembrane space via complex V results in ATP synthesis. Addition of FCCP results in the uncoupling of this process by disrupting the mitochondrial membrane potential and thereby ATP synthesis, while oxygen consumption continues, allowing for the measurement of maximal OCR. Sodium azide (NaN3) is an inhibitor of complexes IV and V, thereby allowing for the measurement of non-mitochondrial respiration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59277/59277fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59277/59277fig2.jpg" /> 
Figure 2: Schematic of steps involved in the measurement of OCR in C. elegans. The five steps involved in the assay setup and run (left) and a timeframe for each step (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59277/59277fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59277/59277fig3.jpg" /> 
Figure 3: Characteristic OCR profile in C. elegans respirometry. The five initial readings show the basal respiration, followed by five readings of maximal and five readings of non-mitochondrial respiration after FCCP and sodium azide injections, respectively. Error bars represent the standard error of measurement (SEM). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59277/59277fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59277/59277fig4.jpg" /> 
Figure 4: Basal respiration in wild type and various sel-12 mutants. Average basal respiration in Day 1 adult age-matched wild type and sel-12 mutant animals. Data compiled from three assay repeats. Error bars represent SEM and **** indicates p &#60;0.0001. p values were calculated using a two-tailed t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59277/59277fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59277/59277fig5.jpg" /> 
Figure 5: Maximal respiration in wild type and various sel-12 mutants. Average maximal respiration in Day 1 adult age-matched wild type and sel-12 mutant animals after FCCP injection. Data compiled from three assay repeats. Error bars represent SEM and **** indicates p &#60;0.0001. p values were calculated using a two-tailed t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59277/59277fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>M9 buffer (1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>1,000 mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 g</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>3 g</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>6 g</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgSO4</td>
			<td>1 mL *add after autoclaving</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Split between 2 bottles: 500 mL each. Autoclave on liquid cycle (15 min exposure)</td>
		</tr>
		<tr>
			<td>Bleach solution (50 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>36 mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>14 mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 N NaOH</td>
			<td>800 &#956;L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Nematode Growth Media (NGM) plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 L</td>
			<td>0.5 L</td>
			<td>0.25 L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>3 g</td>
			<td>1.5 g</td>
			<td>0.75 g</td>
		</tr>
		<tr>
			<td>Bacto-agar</td>
			<td>17 g</td>
			<td>8.5 g</td>
			<td>4.25 g</td>
		</tr>
		<tr>
			<td>Bacto-peptone</td>
			<td>2.5 g</td>
			<td>1.25 g</td>
			<td>0.625 g</td>
		</tr>
		<tr>
			<td>a. Autoclave on liquid cycle (45 min exposure), allow to cool to ~60 &#176;C and then use sterile technique and add the following:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 L</td>
			<td>0.5 L</td>
			<td>0.25 L</td>
		</tr>
		<tr>
			<td>1 M CaCl2</td>
			<td>1 mL</td>
			<td>0.5 mL</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>1 M MgSO4</td>
			<td>1 mL</td>
			<td>0.5 mL</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>1 M KPO4</td>
			<td>25 mL</td>
			<td>12.5 mL</td>
			<td>6.25 mL</td>
		</tr>
		<tr>
			<td>5 mg/mL cholesterol</td>
			<td>1 mL</td>
			<td>0.5 mL</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>b. Swirl to mix thoroughly after each addition. After all additions are made, pour plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Recipes for NGM plates, M9 buffer, and bleach solution. 

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calibration</td>
			<td>Basal</td>
			<td>FCCP&#160;</td>
			<td>Sodium Azide</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Port(s): A</td>
			<td>Port(s): B</td>
		</tr>
		<tr>
			<td>Equilibration: Yes</td>
			<td>Mix: 00:02:00</td>
			<td>Mix: 00:02:00</td>
			<td>Mix: 00:02:00</td>
		</tr>
		<tr>
			<td></td>
			<td>Wait: 00:00:30</td>
			<td>Wait: 00:00:30</td>
			<td>Wait: 00:00:30</td>
		</tr>
		<tr>
			<td></td>
			<td>Measure: 00:02:00</td>
			<td>Measure: 00:02:00</td>
			<td>Measure: 00:02:00</td>
		</tr>
		<tr>
			<td></td>
			<td>Cycles: 5</td>
			<td>Cycles: at least 5</td>
			<td>Cycles: 2&#8722;5</td>
		</tr>
		<tr>
			<td></td>
			<td>Duration: 00:22:30</td>
			<td>Duration: 00:22:30</td>
			<td>Duration: 00:09:00</td>
		</tr>
	</tbody>
</table>
Table 2: Characteristic assay parameters in C. elegans respirometry.

Watch this Scientific Journal Video about Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans at JoVE.com

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

measurement-oxygen-consumption-rates-intact-caenorhabditis

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K Views.  Albany Medical College. Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Video: Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

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