This work was supported by the grants from the National Natural Science Foundation of China (31401013 and 31471010), the Science and Technology Commission of Shanghai Municipality, Shanghai Pujiang Program (14PJ1405900), and Natural Science Foundation of Shanghai (19ZR1446400).

1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster16
<ol>
	<li>Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype.</li>
	<li>Prepare 500 &#181;L of ice-cold extraction buffer containing 20 mM HEPES (pH = 7.2), 1 mM EDTA, and 0.1% triton X-100 in a 1.5 mL test tube for each sample.</li>
	<li>Anesthetize adult flies with CO2 on an anesthesia pad and isolate the desired tissues. To isolate the adult fly thoraxes, for e...

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Metabolic studies using Drosophila as a model must take into consideration the genetic background, diet, and stock maintenance of the flies18. To avoid the effects of different genetic backgrounds on the measurement of citrate synthase activity, different strains of Drosophila should be backcrossed to the control strain for 10 generations. The genetic background of all Drosophila strains used in our experiments is w1118, so we used w1118</sup...

Mitochondria are best known as the power-producing organelles in most eukaryotic organisms, which produce the energy currency, ATP, through the tricarboxylic acid cycle (i.e., Krebs cycle) and oxidative phosphorylation. Mitochondria are also found to play important roles in a lot of other physiological processes, such as regulation of apoptosis1, Ca2+ homeostasis2,3, reactive oxidation species (ROS) generation4, and endoplasmic reticulum (ER)-stress response5. Mitochondrial dysfunction can affect any organ in the body at any age and is a primary cause of metabolic, aging-related6, and neurodegenerative diseases7. Intact mitochondria are mechanistically related to mitochondrial function. Thus, proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function8. Citrate synthase, a rate-limiting enzyme in the first step of the tricarboxylic acid cycle9, is localized in the mitochondrial matrix within eukaryotic cells, and thus can be used as a quantitative marker for the presence of intact mitochondrial mass9,10. Citrate synthase activity can also be used as a normalization factor for intact mitochondrial proteins11,12.
The fruit fly, Drosophila melanogaster, is an excellent model system for studying mitochondrial function, as many molecules and pathways that play pivotal roles in mitochondria are evolutionarily conserved from Drosophila to humans13,14,15. Here, we present a protocol for a fast and simple method for measurement of citrate synthase activity by a colorimetric assay in Drosophila tissue homogenates16 in a 96 well plate format. In the citrate synthase activity assay, citrate synthase in Drosophila tissue homogenate catalyzes the reaction of oxaloacetate with acetyl coenzyme A (acetyl CoA) to form the citrate CoA-SH and H+. CoA-SH subsequently reacts with 5,5&#39;-dithiobis-(2-nitrobenzoic acid) (DTNB) to generate a colored product, 2-nitro-5-thiobenzoate (TNB), which can be easily measured spectrophotometrically at 412 nm. Citrate synthase activity can be reflected by the rate of color production.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>V900477</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA Base)</td>
			<td>Sigma-Aldrich</td>
			<td>V900483</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-CoA</td>
			<td>Sigma-Aldrich</td>
			<td>A2181</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithio-bis-nitrobenzoic acid (DTNB)</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>V900106</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaloacetate</td>
			<td>Sigma-Aldrich</td>
			<td>O4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle</td>
			<td>Sangon</td>
			<td>F619072</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle motor</td>
			<td>Tiangen</td>
			<td>OSE-Y10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>Eon</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein BCA Assay kit</td>
			<td>Beyotime</td>
			<td>P0010S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>WPI</td>
			<td>14124</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sangon</td>
			<td>A110694-0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 presents an example of the kinetic curves for the OD absorbance at 412 nm over time obtained using the citrate synthase activity colorimetric assay to measure the Drosophila thorax tissue homogenates of different genotypes. It is well known that PGC-1&#945; is a master regulator of mitochondrial biogenesis. PGC-1&#945; is functionally conserved between Drosophila and humans. Drosophila RNF34 (dRNF34) is an E3 ubiquitin liga...

Watch this Scientific Journal Video about A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster at JoVE.com

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.

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 ...

Drosophila serves as good model system for studying mitochondrial function. Here we present a protocol for measuring citrate synthase activity in tissue homogenate from adult flies. This protocol does not require isolation of mitochondria.It is fast and simple and it is suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues. Begin by collecting adult flies for each sample, making sure to collect at least triplicate samples for each genotype. Prepare 500 microliters of ice-cold extraction buffer with 20 millimolar HEPES, one millimolar EDTA, and 0.1%triton X-100 in a 1.5-milliliter test tube for each sample.Anesthetize the adult flies with carbon dioxide on an anesthesia pad. To isolate the thoraxes, fix them with a pair of forceps and isolate the fly abdomens by cutting along the border of the thorax and abdomen with a pair of scissors. Collect the fly thoraxes for the citrate synthase activity assay.Transfer 10 adult fly thoraxes to 100 microliters of ice-cold extraction buffer immediately and homogenize them with a pestle on ice. Keep the samples cold by homogenizing for five to 10 seconds on ice, letting them sit on ice for five seconds, then repeating until all tissues are completely homogenized. Aliquot 10 microliters of each homogenized sample into a new tube for protein content measurement, keeping these tubes on ice as well.Add 400 microliters of ice-cold extraction buffer to each remaining sample for a total volume of 500 microliters and pipette up and down gently to mix. For each reaction, add one microliter of diluted cell lysate to 150 microliters of the freshly prepared reaction solution. Mix thoroughly by gently pipetting, making sure to avoid bubble formation, then use a plate reader to measure the absorbance at 412 nanometers every 10 to 30 seconds for four minutes at 25 degrees Celsius.Plot the data as absorbance over time, then calculate the slope for the linear portion of the curve. Finally, divide the reaction rate or slope by the sample protein concentration to normalize the citrate synthase activity. It was hypothesized that knockdown of dRNF34 in Drosophila muscle would increase mitochondrial citrate synthase activity while knockdown of dPGC-1 would reverse this increase.An initial linear enzymatic rate was established for each assay;the slopes of the trendlines represent the maximal reaction rates which are equivalent to the maximal citrate synthase activities of the different genotypes. The maximal citrate synthase activities were calculated from the kinetic curves and normalized by protein concentration. The results demonstrated that the synthase activity of fly thoraxes indeed increased with muscle-specific dRNF34 knockdown and the increase was reversed by muscle-specific dPGC-1 knockdown.When homogenizing the sample, it is important to tip the tube and check the homogenate to make sure that there are no clots and that the tissues in the tube are completely homogenized. After sample collection, all sample treatments should be performed on ice. The citrate synthase activity assay is used to quantify intact mitochondrial mass.Proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function.

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Research

JoVE Journal

Biochemistry

9.8K visualizações.  Shanghai Jiao Tong University Affiliated Sixth People's Hospital. A drosophila serve como um bom sistema de modelo para estudar a função mitocondrial. Aqui apresentamos um protocolo para medir a atividade de sinthase citrato em tecido homogeneado de moscas adultas. Este protocolo não requer isolamento das mitocôndrias.É rápido e simples e é adequado para medir a atividade de sinthase citrato em larvas, células cultivadas e tecidos mamíferos. Comece coletando moscas adultas para cada amostra, certificando-se de coletar pelo menos amostras tri...