Name: small-scale colorimetric assays of intracellular lactate and pyruvate in the nematode caenorhabditis elegans
Uploaded: 2018-10-15T14:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans at JoVE.com

This work was financially supported by a Special Research Grant from Daito Bunka University to Sumino Yanase.

1. Synchronized Culture of C. elegans
<ol>
	<li>Before seeding, culture the Escherichia coli (E. coli) strain OP50 overnight at 37 &#176;C in 300 mL of Luria-Bertani (LB) broth liquid medium. Store the cultured OP50 at -4 &#176;C.
	<ol>
		<li>To make LB broth liquid medium, use 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1.5 mL of 1 N NaOH, and add to 1 L with deionized water. Autoclave. 
		NOTE: OP50 and C. elegans strains are available from the Caenorhabditis Genetics Center (University of Minnesota, St. Paul, MN, USA).</li>
	</ol>
	</li>
	<li>To make nematode growth medium (NGM) agar, use 3 g of NaCl, 2.5 g of peptone, 17 g of agar, and 975 mL of deionized water. Autoclave. Cool to 55 &#176;C, then sterilely add, in order, 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of 5 mg/mL cholesterol in EtOH, and 25 mL of 1 M potassium phosphate pH 6.0 in 90-mm Petri dishes.
	<ol>
		<li>For 1 M potassium phosphate pH 6.0, use 108.3 g of KH2PO4 and 46.6 g of K2HPO4, and add deionized water to 1 L. Autoclave6.</li>
	</ol>
	</li>
	<li>Spread 1-2 mL of the cultured OP50 on the NGM agar plates. To make a thin layer of OP50, incubate the plates overnight at room temperature before adding any nematodes. 
	NOTE: The NGM agar plates inoculated OP50 can be stored at room temperature for 2-3 weeks.</li>
	<li>Add at least 100 worms onto an NGM agar plate with OP50, and culture at 20 &#176;C until the adult stage. At least three plates are required.</li>
	<li>To collect eggs in utero, transfer gravid hermaphrodites from the three NGM agar plates each with 5 mL of S buffer in a 15-mL conical tube, and wash the worms 3 times with 15 mL of S buffer using centrifugation at 300 x g for 30 s at room temperature.
	<ol>
		<li>To make S buffer, use 5.9 g of NaCl and 50 mL of 1 M potassium phosphate pH 6.0, add to 1 L with deionized water. Autoclave6.</li>
	</ol>
	</li>
	<li>Dissolve the worms in an alkaline hypochlorite&#160;solution for axenization and bulk egg isolation (0.5 mL of fresh bleach or equivalent: 5-6% sodium hypochlorite, 0.1 mL of 10 M NaOH, approximately 4.5 mL of S buffer)6, and stand the solution for 10-15 min at room temperature with mixing by inverting. 
	NOTE: Within 15 min, adult worms should dissolve, leaving a hazy solution of eggs liberated from their carcasses (the liberated eggs should be confirmed using a stereoscopic microscope). Instead of 0.1 mL of 10 N NaOH, 0.2 mL of 5 N NaOH can also be used.</li>
	<li>After hypochlorite treatment, wash the egg pellet 3 times with 15 mL of S buffer, and resuspend in 5-6 mL of S buffer. Hatch the released eggs during an overnight incubation at 20 &#176;C in S buffer without E. coli for an age-synchronous culture of L1 stage larvae.</li>
	<li>To determine the approximate number of L1 stage larvae, count the worms using a stereoscopic microscope in 10 &#181;L of S buffer after resuspending the larvae at least 3 times, and calculate the average. Then, transfer the L1 stage larvae to five NGM agar plates with OP50 (1,500-3,000 worms per plate using a 90-mm Petri dish), and culture at 20 &#176;C until they have grown to the young adult stage, when the self-fertilization begins and a few eggs are laid (ordinarily after 3 days).</li>
</ol>
2. Extraction of Cellular Fraction from C. elegans
<ol>
	<li>Collect the young adult stage (5-day-old animals) worms from the five NGM agar plates with S buffer (Figure 1A).
	<ol>
		<li>To select only living worms using the sucrose method6 for flotation on 30% (w/v) sucrose, mix the worms suspended in 3-4 mL of S buffer with an equal volume of ice-cold 60% (w/v) sucrose in a 15-mL conical tube. Spin the tube at 1,500 x g for 15 s at 4 &#176;C and remove the floating worms into a fresh tube by moving them off the wall of the tube with a Pasteur pipette.</li>
	</ol>
	</li>
	<li>Wash the worms 3 times with S buffer by centrifugation at 1,500 x g for 30 s at 4 &#176;C. Check the wet volume of washed worms after centrifugation using a 1,000 &#181;L micropipette tip (Figure 1B).</li>
	<li>Add the washed worms to an equal volume of ice-cold 10% (w/v) trichloroacetic acid (TCA; final concentration of 5%) for protein precipitation (Figure 1C). Instead of TCA, perchloric acid (PCA) or metaphosphoric acid can be used.</li>
	<li>Homogenize the worms with the precipitant using 40 strokes of a pestle in a Teflon homogenizer (Potter-Elvehjem tissue grinder) with rotation at up to 1,300 rpm on ice.</li>
	<li>Transfer the homogenate into a fresh 1.5-mL microtube with a Pasteur pipette, and sonicate using an ultrasonic homogenizer for 3 min (3 times of 1 min) with a 20% duty cycle on ice.</li>
	<li>Clarify the homogenate by centrifugation at 8,000 x g for 10 min at 4 &#176;C. Neutralize the supernatants with 4 M KOH (0.25 volume to 10% TCA) for 20 min on ice, and centrifuge at 8,000 x g for 10 min at 4 &#176;C. The supernatant (as a test sample) can be stored at -80 &#176;C until the following assays.</li>
</ol>
3. Lactate Assay Using a Colorimetric Assay Kit
<ol>
	<li>Measure the concentration of lactate in the test samples using a colorimetric assay kit (Table of Materials). Carry out duplex examinations for the test samples. Add 5 or 10 &#181;L of the test samples to a 96-well plate and adjust the volume to 50 &#181;L per well with the Lactate Assay Buffer provided with the kit.</li>
	<li>For the lactate standard curve, dilute 100 mM L(+)-Lactate Standard to 1 mM with Lactate Assay Buffer. Add 0, 2, 4, 6, 8, and 10 &#181;L of the 1 mM L(+)-Lactate Standard, which is provided with the kit, into a series of wells.</li>
	<li>Add 50 &#181;L of Reaction Mix (containing 46:2:2 of Lactate Assay Buffer, Lactate Enzyme Mix and Lactate Probe in DMSO, anhydrous; all reagents are provided with the kit) or Background Control Mix (containing 48:2 of Lactate Assay Buffer and Lactate Probe) into each well and incubate at room temperature for 30-60 min while the samples are protected from light.</li>
	<li>Measure the absorbance of each well at 570 nm using a microplate reader and subtract the absorbance of the Background Control Mix from the absorbance of the Reaction Mix.</li>
	<li>Plot the lactate standard curve. Calculate the lactate concentrations of the test samples from the lactate standard curve.</li>
</ol>
4. Pyruvate Assay Using a Colorimetric Assay Kit
<ol>
	<li>Measure the concentration of pyruvate in the supernatants (test samples) using a colorimetric assay kit (Table of Materials). Carry out duplex examinations for the test samples. Add 10 &#181;L of the test samples and 90 &#181;L of Working Reagent (containing 94:1 of Enzyme Mix and Dye Reagent, which are provided with the kit) into a 96-well plate and incubate at room temperature for 30 min while the samples are protected from light.</li>
	<li>Measure the absorbance of each well at 570 nm using a microplate reader.</li>
	<li>Plot the pyruvate standard curve. Calculate the pyruvate concentrations of the test samples from the pyruvate standard curve.</li>
</ol>
5. Protein Assay for Normalization with Protein Content
<ol>
	<li>Measure the protein concentration in the supernatants (test samples) using a colorimetric assay kit (Table of Materials). However, this step is not limited to an assay kit, and other approaches can be utilized to measure the protein concentration.</li>
	<li>Normalize the values of lactate and pyruvate concentrations among the test samples using each total protein concentration. 
	Note: Protein concentration in the test samples is sufficiently detected even after protein precipitation and is utilized for the normalization step among samples.</li>
</ol>

<ol>
	<li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+cells.">On the origin of cancer cells.</a> Science. 123, 309-314 (1956).</li><li>Matoba, S., 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=p53+regulates+mitochondrial+respiration.">p53 regulates mitochondrial respiration.</a> Science. 312, 1650-1653 (2006).</li><li>Yanase, S., Suda, H., Yasuda, K., Ishii, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+p53/CEP-1+is+associated+with+lifespan+extension+through+an+age-related+imbalance+in+the+energy+metabolism+of+C.+elegans.">Impaired p53/CEP-1 is associated with lifespan extension through an age-related imbalance in the energy metabolism of C. elegans.</a> Genes to Cells. 22 (12), 1004-1010 (2017).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> WormBook. , (2007).</li><li>Hulme, S. E., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+the+worm:+Caenorhabditis+elegans+as+a+platform+for+integrating+chemical+and+biological+research.">Chemistry and the worm: Caenorhabditis elegans as a platform for integrating chemical and biological research.</a> Angewandte Chemie International Edition. 50, 4774-4807 (2011).</li><li>Lewis, J. A., Fleming, J. T., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+culture+methods.">Basic culture methods.</a> Methods in Cell Biology, Volume 48, Caenorhabditis elegans: Modern Biological Analysis of an Organism. , 3-29 (1995).</li><li>Senoo-Matsuda, N., 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=A+defect+in+the+cytochrome+b+large+subunit+in+complex+II+causes+both+superoxide+anion+overproduction+and+abnormal+energy+metabolism+in+Caenorhabditis+elegans.">A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans.</a> The Journal of Biological Chemistry. 276 (45), 41553-41558 (2001).</li><li>Butler, J. A., Mishur, R. J., Bhaskaran, S., Rea, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+signature+for+long+life+in+the+Caenorhabditis+elegans+Mit+mutants.">A metabolic signature for long life in the Caenorhabditis elegans Mit mutants.</a> Aging Cell. 12, 130-138 (2013).</li><li>Marbach, E. P., Weil, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enzymatic+measurement+of+blood+lactate+and+pyruvate:+Use+and+significance+of+metaphosphoric+acid+as+a+common+precipitant.">Rapid enzymatic measurement of blood lactate and pyruvate: Use and significance of metaphosphoric acid as a common precipitant.</a> Clinical Chemistry. 13 (4), 314-325 (1967).</li><li>Mishur, R. J., 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+metabolites+extend+lifespan.">Mitochondrial metabolites extend lifespan.</a> Aging Cell. 15, 336-348 (2016).</li></ol>

The authors declare that they have no competing financial interests.

When utilizing these colorimetric assay kits, the most critical step in sample extraction to detect cellular lactate and pyruvate accurately in C. elegans is the process of protein precipitation during homogenization (Figure 1). It is not strictly necessary to use a Teflon homogenizer, as other homogenizers (e.g., Dounce and tapered tissue grinders, or bead mills) are also suited to the small-scale extraction of worms. We did not detect cellular lactate and pyruvate in test samples that were extracted using a homogenizer before protein precipitation. Furthermore, both lactate and pyruvate levels decreased drastically in the cytosolic fraction separated using ultracentrifugation instead of protein precipitation (Table 1). These suggest that the sample extraction is crucial to detect the cellular metabolites in biochemical methodology using a nematode C. elegans. For protein precipitation, not only TCA but also several other precipitants (PCA or metaphosphoric acid) can be utilized to the biochemical methodology7,9. According to a previous report, the use of TCA resulted in the disappearance of approximately 12% of NADH in the enzymatic measurement of blood lactate and pyruvate. Therefore, the authors concluded that 5% metaphosphoric acid should be selected as the protein precipitant for both lactate and pyruvate when using enzymatic assays9. It suggests that TCA partially denatures proteins including many enzymes.However, we had no problems regarding the protein precipitants when using colorimetric assays to detect cellular lactate and pyruvate.
In this study, we used commercial colorimetric assay kits for the quantitative determination of lactate and pyruvate (Figure 2 and&#160;Figure 3). Their sensitivities and sample sizes were superior in comparison with previous assay kits7; therefore, we prepared the test samples using a smaller scale extraction and reported the differences in the cellular lactate and pyruvate concentrations in C. elegans3. We detected an age-related decrease in lactate and consequent L/P ratio in wild-type C. elegans and found that the p53/CEP-1 has an important role in the age-related alteration of energy metabolism through the activation of its transcriptional targets2,3. Thus, the analysis of energy metabolism in nematode C. elegans advantageously proceeds due to using the improved colorimetric assay kits, at least in part.
As shown in Figure 3B, lactate concentration below 2 mM tend to be indeed larger in this colorimetric assay (not the fluorometric assay). Therefore, the fluorometric assay may be suitable for a more accurate measurement of lactate. The capabilities of these assay kits support both colorimetric and fluorometric assays, so that the assay is selected in response to the appliance in laboratory of the user. It is still more expensive to use the present commercial colorimetric assay kits to determine quantitatively the concentration of cellular metabolites. However, trials can be adequately performed in smaller laboratories using common instruments and a spectrophotometric plate reader.
Hitherto, there are relatively few reports using conventional biochemical approaches in a model organism C. elegans, which typically need the large-scale culture of worms, compared with genetic studies5. However, the recent remarkable progress of biological assay systems (e.g., improvement of the sensitivity and stability of assay kits) makes studies using the nematode C. elegans, which is small and consists of fewer than 1,000 cells, more attractive and performed easily in the laboratory. Henceforth, effective metabolomic analysis using mass spectrometry (MS) and gas chromatography/mass spectrometry (GC/MS) can help to reduce certain problems, such as large-scale culture, analytical sensitivity, and specificity, and will help to elucidate the role of cellular metabolism in aging and human diseases including cancer10. Looking ahead, easier methodology, such as colorimetric assay kits, would be effective as a screening tool prior to more precise instrumental analyses.

Lactate and pyruvate concentrations are widely regarded as intermediates of energy metabolism, and are related to the states of glycolysis, tricarboxylic acid (TCA) cycle, and electron transport chain in the cells of aerobic organisms. A series of reactions in glycolysis oxidize glucose to pyruvate, which lies at a metabolic crossroads and can be converted to carbohydrates through gluconeogenesis, to fatty acids and energy metabolism through acetyl-CoA, and to the amino acid alanine. The TCA cycle occurs under the presence of sufficient dissolved oxygen and is fundamental for the conversion of glucose to energy. Especially, the alteration of secondary metabolism is an interesting phenomenon in which glycolysis is used predominantly for energy production and aerobic mitochondrial respiration, which involves the TCA cycle and electron transport chain, is downregulated in mammalian cancer cells1,2. We showed recently that the lactate levels and the consequent lactate/pyruvate (L/P) ratio decreased during aging in the model organism Caenorhabditis elegans (C. elegans). Likewise, we found that the mammalian tumor suppressor p53 ortholog CEP-1 in C. elegans has an important role in the age-related alterations of energy metabolism through the activation of its transcriptional targets3.
In biological assays, such as the measurement of lactate and pyruvate concentrations in cells, the sensitivity, accuracy, sample size, and incubation time of colorimetric/fluorimetric assay kits have been improved dramatically. Owing to technological innovations, we are now able to analyze various metabolites and intermediate metabolites without the large-scale culture of C. elegans, which is difficult given its small size. In general, the sensitivity of a colorimetric assay is an order of magnitude smaller than that of a fluorimetric assay; however, the colorimetric approach is more suitable in the setting of common laboratories. Furthermore, an extraction technique containing homogenization and protein precipitation is crucial for the quantitative determination of lactate and pyruvate concentrations in C. elegans cells because this nematode is enclosed in an exoskeleton called the cuticle, unlike mammalian cultured cell lines4,5. Here, we describe a protocol to analyze lactate and pyruvate concentrations using commercial colorimetric assay kits including tips for sample extraction from C. elegans.

<table><tbody><tr><td>Lactate Colorimetric/Fluorimetric Assay kit&amp;nbsp;</td><td>BioVision</td><td>#K607-100</td><td>colorimetric/fluorimetric&lt;br/&gt; 100 assays; Store at -20oC</td></tr><tr><td>EnzyChrom Pyruvate&lt;br/&gt; Assay Kit</td><td>BioAssay&lt;br/&gt; Systems</td><td>#EPYR-100</td><td>colorimetric/ fluorometric&lt;br/&gt; 100 assays; Store at -20oC</td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>#23225</td><td>colorimetric assay; store at&lt;br/&gt; &amp;nbsp;room temperature</td></tr><tr><td>Trichloroacetic Acid</td><td>Wako Pure Chemical</td><td>#207-04955</td><td>store at room temperature</td></tr><tr><td>Teflon homogenizer&amp;nbsp;</td><td>Iwaki/Pyrex</td><td>#358034 (Wheaton)</td><td>Instead of Iwaki/Pyrex,&lt;br/&gt; available by Wheaton</td></tr></tbody></table>

small-scale colorimetric assays of intracellular lactate and pyruvate in the nematode caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Using the colorimetric assays for the quantitative determination of lactate and pyruvate concentrations, we showed the accuracy of these assays compared with previous reports in C. elegans7,8. Here, the process of protein precipitation during sample extraction was the most crucial step to generate accurate values. For protein precipitation, common precipitants (e.g., TCA, PCA, or metaphosphoric acid) can be used to prepare the test samples (Figure 1). In C. elegans, however, it was necessary to perform protein precipitation during homogenization of the worms (Table 1). In addition to accuracy, these assays were sufficiently sensitive to measure lactate and pyruvate concentrations in small-scale samples and were able to detect them in a short period of time (the reactive incubation time of both assays is at least 30 min) (Figure&#160;2-3). Actually, we presented the data in a recent report3. Thus, we could detect age-related metabolic alterations indicating that cellular lactate levels and the consequent L/P ratio decreased during aging, and we also showed different energy metabolism in a cep-1 mutant3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57807/57807fig1.jpg" /> 
Figure 1.&#160;Process for the extraction of cellular metabolites. (A) 1,500-3,000 worms were placed on an NGM agar plate (90-mm Petri dish). Extraction from worms on five plates was sufficient for the colorimetric assays. (B)&#160;Worms collected from the five plates of 3,000 and 1,500 worms per plate are indicated on the left and right side of panel, respectively. Both the wet volumes in each 15-mL tube are &#60; 0.5 mL, which were sufficient for detection. (C) Protein precipitation using 10% TCA during homogenization of the worms. Adding the worms into ice-cold 10% TCA in a homogenizer has to be performed before the worms are homogenized. Otherwise, cellular lactate and pyruvate cannot be detected in test samples using a colorimetric assay kit (data are shown in Table 1). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57807/57807fig1large.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/57807/57807fig2.jpg" /> 
Figure 2.&#160;Colorimetric pigmentation patterns of the lactate standards using a colorimetric assay kit. (A)&#160;Wells of the 96-well plate used in the colorimetric assay, and a dilution series of the L(+)-lactate standard. Increasing the lactate concentration resulted in a more intense pink color. BG indicates the background of a lactate probe against the dilution series. (B)&#160;Lactate standard curve for the colorimetric assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57807/57807fig2large.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/57807/57807fig3.jpg" /> 
Figure 3.&#160;Colorimetric pigmentation patterns of the pyruvate standards using a colorimetric assay kit. (A)&#160;Wells of the 96-well plate using the colorimetric assay, and dilution series of pyruvate standard. Increasing of the pyruvate concentration resulted in increased light pink coloring. (B)&#160;Pyruvate standard curve for the colorimetric assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57807/57807fig3large.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>Test Samples</td>
			<td>Lactate (mM)</td>
			<td>Pyruvate (mM)</td>
		</tr>
		<tr>
			<td>Protein-precipitated samples during homogenization</td>
			<td>3.07 &#177; 0.94</td>
			<td>0.22 &#177; 0.08</td>
		</tr>
		<tr>
			<td>Protein-precipitated samples after homogenization</td>
			<td>ND</td>
			<td>ND</td>
		</tr>
		<tr>
			<td>Intact cytosolic fraction after homogenization and ultracentrifugation*&#160;</td>
			<td>1.12</td>
			<td>0.06</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Effects of protein precipitation on different timings for the detection of cellular lactate and pyruvate in 5-day-old animals of wild-type N2. Data indicate means + standard deviation (SD) of at least three determinations. ND indicates not detected. *The extraction process was performed preliminarily using ultracentrifugation without protein precipitation.

Watch this Scientific Journal Video about Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans at JoVE.com

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

small-scale-colorimetric-assays-intracellular-lactate-pyruvate

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.9K Views.  Daito Bunka University. Dit onderzoek kan helpen bij het beantwoorden van vragen in hen biochem tellen en methodologie veld, zoals een analyse van de ontwikkelingsrechten in aaltjes. Het belangrijkste voordeel van deze techniek is eiwitneerslag in extraductie en drank en homogenisatie van de wormen. Start dit experiment door het kweken van E.coli OP50 stam in 300 milliliter LB bouillon vloeibaar medium op 37 graden Celsius 's nachts.Bewaar succesvolle OP50 op vier graden Celsius. Pipette twee milliliters van ...

Video: Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Developmental Biology

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Neuroscience

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Biology

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

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.

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.

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

Evaluating Metabolism and Aging Pattern in Caenorhabditis elegans

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

Author Spotlight: Exploring Metabolic and Aging Processes in C. elegans Using Low-Cost, High-Impact Assays

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences ...

Colorimetric Assay for Quantification of Lactate in Test Samples

Source: Yanase, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57807">Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans.</a> J. Vis. Exp. (2018).
This video describes a colorimetric assay for lactate measurement in cell lysates by using the lactate dehydrogenase enzyme. This enzyme converts lactate into pyruvate, along with the conversion of NAD to NADH that reacts with tetrazolium dyes and gives a colored product that can be measured spectrophotometrically to quantitate the lactate.

Source: Yanase, S., et al. Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms

Source: Yanase, S. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57807">Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode&#160;Caenorhabditis elegans.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes a colorimetric assay for quantitatively measuring pyruvate in the cellular fractions of Caenorhabditis elegans. Utilizing the hydrogen peroxide produced from the enzymatic reaction of pyruvate oxidase with cellular pyruvate, a chromogenic dye is oxidized to produce a colored product, measured using colorimetry to determine the pyruvate concentration.

Source: Yanase, S. et al. Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode&#160;Caenorhabditis elegans.&#160;J. Vis. ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video