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...

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 border="0" cellpadding="0" cellspacing="0" style="width:696px;" width="696">
	<tbody>
		<tr height="62">
			<td height="62" style="height:62px;width:247px;">Lactate Colorimetric/Fluorimetric Assay kit&#160;</td>
			<td style="width:100px;">BioVision</td>
			<td style="width:136px;">#K607-100</td>
			<td style="width:213px;">colorimetric/fluorimetric 
			100 assays; Store at -20oC</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">EnzyChrom Pyruvate 
			Assay Kit</td>
			<td style="width:100px;">BioAssay 
			Systems</td>
			<td style="width:136px;">#EPYR-100</td>
			<td style="width:213px;">colorimetric/ fluorometric 
			100 assays; Store at -20oC</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">BCA Protein Assay Kit</td>
			<td style="width:100px;">Thermo Scientific</td>
			<td style="width:136px;">#23225</td>
			<td style="width:213px;">colorimetric assay; store at 
			&#160;room temperature</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Trichloroacetic Acid</td>
			<td style="width:100px;">Wako Pure Chemical</td>
			<td style="width:136px;">#207-04955</td>
			<td>store at room temperature</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:247px;">Teflon homogenizer&#160;</td>
			<td style="width:100px;">Iwaki/Pyrex</td>
			<td style="width:136px;">#358034 (Wheaton)</td>
			<td style="width:213px;">Instead of Iwaki/Pyrex, 
			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 ...

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

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Biochemistry

6.9K Views.  Daito Bunka University. 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.

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 ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...