eoe sub articles

EoE Sub Articles

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. Autoclave.</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, and add to 1 L with deionized water. Autoclave.</li>
	</ol>
	</li>
	<li>Dissolve the worms in an alkaline hypochlorite 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), 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 method 6 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. 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>

<table><tbody><tr><td>EnzyChrom Pyruvate Assay Kit</td><td>BioAssay Systems</td><td>#EPYR-100</td><td>colorimetric/ fluorometric 100 assays; Store at -20&amp;deg;C</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, available by Wheaton</td></tr></tbody></table>

pyruvate assay for estimation of pyruvate levels in cellular fractions of worms

Source: Yanase, S. et al. <a href="https://app.jove.com/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.

<img alt="Figure 1" src="/files/ftp_upload/21320/21320_Figure1.jpg" /> 
Figure 1. 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) 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.

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

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

Cellular levels of pyruvate &#8212; a key intermediate in energy-producing biochemical pathways &#8212; can be estimated using enzyme-based colorimetric assays.
To determine pyruvate levels in the cells of Caenorhabditis elegans &#8212; a non-parasitic nematode &#8212; take a cellular fraction prepared from adult worms containing pyruvate. Treat the solution, removing cellular proteins, and collect the clarified cellular fraction &#8212; the test sample &#8212; containing pyruvate. The absence of cellular proteins ensures an accurate determination of pyruvate levels.
Start by adding a working reagent containing the enzymes pyruvate oxidase and horseradish peroxidase, and a chromogenic dye. Incubate the mixture in the dark for an adequate duration.
During incubation, pyruvate oxidase reacts with pyruvate &#8212; its substrate &#8212; generating hydrogen peroxide as a byproduct. The absence of light prevents the spontaneous decomposition of hydrogen peroxide.
Horseradish peroxidase catalyzes the reaction between the hydrogen peroxide and the chromogenic dye, producing a colored product. The intensity of the colored reaction product is directly proportional to the pyruvate concentration in the sample.
Upon completion of the incubation period, place the sample inside a plate reader and measure the absorbance. Generate a standard curve using a serial dilution with known pyruvate concentrations.
Plot the measured absorbance value of the test sample on the curve to determine the pyruvate concentration in the test sample.

pyruvate-assay-for-estimation-pyruvate-levels-cellular-fractions

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

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

Video: Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms - Concept

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

JoVE Journal

Cancer Research

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 ...

Biochemistry

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...

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

Transcript

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

Hyperpolarized ¹³C Metabolic Magnetic Resonance Spectroscopy and Imaging

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method