Name: measurement of oxygen consumption rates in intact caenorhabditis elegans
Uploaded: 2019-02-23T15:00:00
Description: Watch this Scientific Journal Video about Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans at JoVE.com

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 le...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Morphology+and+Fundamental+Parameters+of+the+Mitochondrial+Respiratory+Chain+Are+Altered+in+Caenorhabditis+elegans+Strains+Deficient+in+Mitochondrial+Dynamics+and+Homeostasis+Processes.">Mitochondrial Morphology and Fundamental Parameters of the Mitochondrial Respiratory Chain Are Altered in Caenorhabditis elegans Strains Deficient in Mitochondrial Dynamics and Homeostasis Processes.</a> PLoS One. 10, e0130940 (2015).</li><li>Ryu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urolithin+A+induces+mitophagy+and+prolongs+lifespan+in+C.+elegans+and+increases+muscle+function+in+rodents.">Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents.</a> Nature Medicine. 22, 879-888 (2016).</li><li>Perry, C. 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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, especia...

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 compar...

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 ...

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