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 example, fix the fly thoraxes by a pair of forceps, and then isolate the fly abdomens by cutting along the border of the thorax and abdomen using a pair of scissors. Collect the fly thoraxes for the muscle citrate synthase activity assay. 
	NOTE: Determine the fresh weight of tissue at this step if using it to normalize citrate synthase activity.</li>
	<li>Transfer 10 adult fly thoraxes to 100 &#181;L of ice-cold extraction buffer immediately and homogenize with a pestle on ice. To keep the samples ice-cold, homogenize the samples for 5-10 s on ice, then have the samples sit on ice for 5 s, and repeat until all the tissues in the tube are homogenized completely. 
	NOTE: Tape the tube, check the homogenates to make sure that there are no clots in the homogenates and that the tissues in the tube are homogenized completely. All sample treatments should be performed on ice.</li>
	<li>Take 10 &#181;L of each homogenized sample in a new tube for protein content measurement. Keep the samples for protein measurement on ice. 
	NOTE: The protein samples can be frozen and stored at -80 &#176;C for later analysis. The protein concentrations serve as an internal parameter for normalization of citrate synthase activities.</li>
	<li>Add 400 &#181;L of ice-cold extraction buffer to each remaining sample to make a total volume of 500 &#181;L. Mix well by gently pipetting up and down at least 5x, avoiding bubble formation. For each reaction, add 1 &#181;L of diluted cell lysate to 150 &#181;L of the freshly prepared reaction solution (20 mM Tris-HCl (pH 8.0), 0.1 mM DTNB, 0.3 mM acetyl CoA, 1 mM oxaloacetic acid). Mix thoroughly and immediately by gently pipetting, avoiding bubble formation. Measure the absorbance at 412 nm every 10-30 s for 4 min at 25 &#176;C using a plate reader capable of measuring absorbance at 412 nm at minimum intervals of 10 s. 
	NOTE: Change the tip before pipetting a different reagent and avoid forming bubbles in the wells. Incomplete mixing of the reagents may cause variations in the measurements. The citrate synthase activity assay should be done at room temperature immediately after the samples are collected, as this assay is used to quantify intact mitochondrial mass. The reaction buffer should be prepared immediately before use and should not be stored.</li>
	<li>Plot data as optical density (OD) absorbance (abs) (Y-axis) versus time (in minutes) (X-axis). Then calculate the slope for the linear portion of the curve, where the reaction rate is 
	 
	<img src="/files/ftp_upload/59454/59454eq1.jpg" /> 
	 
	Divide the value by the sample protein concentration to normalize the citrate synthase activity. The citrate synthase activity is calculated as 
	 
	<img src="/files/ftp_upload/59454/59454eq2.jpg" /> 
	 
	NOTE: The sample protein concentration can be measured by a protein concentration assay kit.</li>
</ol>

<ol>
	<li>Yee, C., Yang, W., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intrinsic+apoptosis+pathway+mediates+the+pro-longevity+response+to+mitochondrial+ROS+in+C.+elegans.">The intrinsic apoptosis pathway mediates the pro-longevity response to mitochondrial ROS in C. elegans.</a> Cell. 157 (4), 897-909 (2014).</li><li>Sarasija, S., Norman, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gamma-Secretase+Independent+Role+for+Presenilin+in+Calcium+Homeostasis+Impacts+Mitochondrial+Function+and+Morphology+in+Caenorhabditis+elegans.">A gamma-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans.</a> Genetics. 201 (4), 1453-1466 (2015).</li><li>Oxenoid, K., 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=Architecture+of+the+mitochondrial+calcium+uniporter.">Architecture of the mitochondrial calcium uniporter.</a> Nature. 533 (7602), 269-273 (2016).</li><li>Hekimi, S., Wang, Y., Noe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ROS+and+the+Effectors+of+the+Intrinsic+Apoptotic+Pathway+in+Aging+Cells:+The+Discerning+Killers!.">Mitochondrial ROS and the Effectors of the Intrinsic Apoptotic Pathway in Aging Cells: The Discerning Killers!.</a> Frontiers in Genetics. 7, 161 (2016).</li><li>Kim, H. E., 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=Lipid+Biosynthesis+Coordinates+a+Mitochondrial-to-Cytosolic+Stress+Response.">Lipid Biosynthesis Coordinates a Mitochondrial-to-Cytosolic Stress Response.</a> Cell. 166 (6), 1539-1552 (2016).</li><li>Wang, Y., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+longevity+in+animals:+Untangling+the+knot.">Mitochondrial dysfunction and longevity in animals: Untangling the knot.</a> Science. 350 (6265), 1204-1207 (2015).</li><li>Ray, A., Martinez, B. A., Berkowitz, L. A., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction,+oxidative+stress,+and+neurodegeneration+elicited+by+a+bacterial+metabolite+in+a+C.+elegans+Parkinson's+model.">Mitochondrial dysfunction, oxidative stress, and neurodegeneration elicited by a bacterial metabolite in a C. elegans Parkinson's model.</a> Cell Death &amp; Disease. 5, e984 (2014).</li><li>Tronstad, K. 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=Regulation+and+quantification+of+cellular+mitochondrial+morphology+and+content.">Regulation and quantification of cellular mitochondrial morphology and content.</a> Current Pharmaceutical Design. 20 (35), 5634-5652 (2014).</li><li>Wiegand, G., Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrate+synthase:+structure,+control,+and+mechanism.">Citrate synthase: structure, control, and mechanism.</a> Annual Review of Biophysics and Biophysical Chemistry. 15, 97-117 (1986).</li><li>Lopez-Lluch, G., 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=Calorie+restriction+induces+mitochondrial+biogenesis+and+bioenergetic+efficiency.">Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (6), 1768-1773 (2006).</li><li>MacInnis, M. 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=Superior+mitochondrial+adaptations+in+human+skeletal+muscle+after+interval+compared+to+continuous+single-leg+cycling+matched+for+total+work.">Superior mitochondrial adaptations in human skeletal muscle after interval compared to continuous single-leg cycling matched for total work.</a> Journal of Physiology. 595 (9), 2955-2968 (2017).</li><li>Gillen, J. B., 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=Twelve+Weeks+of+Sprint+Interval+Training+Improves+Indices+of+Cardiometabolic+Health+Similar+to+Traditional+Endurance+Training+despite+a+Five-Fold+Lower+Exercise+Volume+and+Time+Commitment.">Twelve Weeks of Sprint Interval Training Improves Indices of Cardiometabolic Health Similar to Traditional Endurance Training despite a Five-Fold Lower Exercise Volume and Time Commitment.</a> PLoS One. 11 (4), e0154075 (2016).</li><li>Mukherjee, S., Basar, M. A., Davis, C., Duttaroy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+functional+similarities+and+divergences+between+Drosophila+Spargel/dPGC-1+and+mammalian+PGC-1+protein.">Emerging functional similarities and divergences between Drosophila Spargel/dPGC-1 and mammalian PGC-1 protein.</a> Frontiers in Genetics. 5, 216 (2014).</li><li>Mukherjee, S., Duttaroy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spargel/dPGC-1+is+a+new+downstream+effector+in+the+insulin-TOR+signaling+pathway+in+Drosophila.">Spargel/dPGC-1 is a new downstream effector in the insulin-TOR signaling pathway in Drosophila.</a> Genetics. 195 (2), 433-441 (2013).</li><li>Diop, S. B., 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=PGC-1/Spargel+Counteracts+High-Fat-Diet-Induced+Obesity+and+Cardiac+Lipotoxicity+Downstream+of+TOR+and+Brummer+ATGL+Lipase.">PGC-1/Spargel Counteracts High-Fat-Diet-Induced Obesity and Cardiac Lipotoxicity Downstream of TOR and Brummer ATGL Lipase.</a> Cell Reports. 10 (9), 1572-1584 (2015).</li><li>Rera, M., 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=Modulation+of+longevity+and+tissue+homeostasis+by+the+Drosophila+PGC-1+homolog.">Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog.</a> Cell Metabolism. 14 (5), 623-634 (2011).</li><li>Wei, P., 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=RNF34+modulates+the+mitochondrial+biogenesis+and+exercise+capacity+in+muscle+and+lipid+metabolism+through+ubiquitination+of+PGC-1+in+Drosophila.">RNF34 modulates the mitochondrial biogenesis and exercise capacity in muscle and lipid metabolism through ubiquitination of PGC-1 in Drosophila.</a> Acta Biochimica et Biophysica Sinica (Shanghai). 50 (10), 1038-1046 (2018).</li><li>Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68 (1), 105-115 (2014).</li><li>Bosco, G., 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=Effects+of+oxygen+concentration+and+pressure+on+Drosophila+melanogaster:+oxidative+stress,+mitochondrial+activity,+and+survivorship.">Effects of oxygen concentration and pressure on Drosophila melanogaster: oxidative stress, mitochondrial activity, and survivorship.</a> Archives of Insect Biochemistry and Physiology. 88 (4), 222-234 (2015).</li></ol>

The authors declare no conflicts of interest.

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 as a control. Generally, we think w1118 is a good control, as it is the genetic background for most Drosophila strains and is much easier to backcross than Canton-S. The diet and stock maintenance must be exactly the same for all the experimental groups. In our experiments, flies are normally maintained at 25 &#176;C and 50-60% relative humidity. The sample preparation step should also be performed with caution. The crosses that are set up for the experiment should be in a similar and appropriate scale. To set up a cross, 3-4 female virgin adult flies are kept with 2-3 male adult flies in a 4 cm diameter, 15 cm high vial supplied with fresh food. The food recipe can vary (e.g., high fat diet, normal diet, high sugar diet), depending on the experimental design. Two days later, the parental generation of flies is transferred to a new vial with fresh food. Caring for the larvae hatched in the food includes adding some water if necessary. When the filial generation of flies starts to hatch, the vials are flipped every 24 h, the flies of the desired genotype are collected in a new vial for the experiment, and the hatch date of the flies is marked on each vial. Approximately 20-30 collected flies of the same genotype, gender, and age are kept in a vial with food. The collected flies must be transferred to fresh vials with appropriate food every two days until the experiments are performed. To avoid the effect of age on citrate synthase activity, the flies of the different groups tested should hatch on the same day. In addition, the gender of the flies should be matched. Usually the body size of females is larger than that of males, thus the protein concentration of female bodies is higher than that of male bodies.
The citrate synthase activity assay can also be used for measuring the citrate synthase activity in larvae. To this end, each sample can consist of five wandering third instar larvae. The third instar larvae can be distinguished by a dark orange ring at the tip of their posterior spiracles, which is lacking or weakly present in the second instar larvae.
In step 6, the dilution ratios of samples may vary for different samples. For the samples that are first measured, several dilution ratios have to be tried to determine an appropriate dilution ratio to establish a linear enzymatic rate in the assay. The total measuring time for the maximal enzyme activity that establishes a linear enzymatic rate in the assay varies depending on the enzyme-substrate ratio. If the substrate is extremely overdosed compared to the enzyme, then the enzyme activity or the reaction rate reaches maximal, which allows the establishment of a linear enzymatic rate in the assay. As the reaction goes on, the amount of the substrate decreases, and the enzyme activity or the reaction rate slows down. If a linear enzymatic rate cannot be plotted, which means that the substrate is not overdosed compared to the enzyme, further dilute the tissue homogenates with reaction solution or shorten the interval time for the 412 nm readings until a linear enzymatic rate plot is established. The interval time for the 412 nm reading should be no more than 30 s. The duration for the 412 nm reading is based on the reaction rate and time. The spectrophotometer reading should stop when no further color change is observed for all reactions.
The citrate synthase activity can be normalized by the sample protein concentration, sample fresh weight, or cell number. The sample fresh weight can be measured before homogenizing as in step 3. The cell number can also be used to normalize the lysate of cultured cells, but the cells must be completely lysed. DNA content is not a good option for an internal control, as the DNA extraction procedure may introduce variations in the samples, particularly for samples containing a small amount of DNA.
If no color change is observed after the reaction, several different troubleshooting steps can be taken: 
1. Check the sample preparation step, making sure to handle the samples properly, keep the samples on ice, and avoid multiple freeze-thaw cycles. 
2. Try to use a higher protein concentration, because some samples might have much lower expression levels of citrate synthase that are undetectable by the citrate synthase activity assay. 
3. Note that some commercially available kits for citrate synthase activity assay cannot be used for Drosophila, because these kits determine mitochondrial citrate synthase activity based on immunocapture of mammalian citrate synthase and the mammalian citrate synthase antibody may not recognize the Drosophila citrate synthase.
Likewise, if there is a discrepancy between samples: 
1. Make sure there are no bubbles in the wells. 
2. Examine whether this is caused by poor pipetting technique.
In contrast to measuring citrate synthase activity by a colorimetric assay in purified mitochondria, which requires almost 200 flies for the purification of mitochondria of each genotype19, we present a protocol for a fast and simple assay for measurement of citrate synthase activity by a colorimetric method in tissue homogenate or in whole cell extracts16. For each sample, only ten flies hatched on the same day are needed; for triplicate samples of each genotype, 30 flies are needed. The protocol described is applicable not only to Drosophila but also to other systems, such as cultured cells and mammalian tissues.
Besides the citrate synthase activity assay, other methods can be used to quantify mitochondrial mass. Compared to other commonly used quantitative methods of mitochondria, such as measurement of mitochondrial DNA content by qPCR and quantification of mitochondrial proteins by Western blotting, which detect all the mitochondria regardless of their function, the citrate synthase activity assay is used to quantify the presence of intact mitochondrial mass. Unlike the mitochondrial respiration assay, which needs mitochondrial purification and specialized assay equipment16, the citrate synthase activity assay allows researchers to carry out rapid measurements by spectrophotometry with simple sample preparation, such as whole cell extract or tissue homogenate, and no need for mitochondrial isolation. Thus, the citrate synthase activity assay is a rapid and economical assay for quantification of intact mitochondrial mass.

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 ligase for Drosophila PGC-1&#945;, dPGC-1, and promotes dPGC-1 protein degradation17. Transmission electron microscopy and mitochondrial DNA qPCR have shown that knockdown of dRNF34 in Drosophila muscle increases mitochondrial content, which is suppressed by knockdown of dPGC-117. Based on these results we hypothesized that knockdown of dRNF34 in Drosophila muscle would increase mitochondrial citrate synthase activity, which should be reversed by knockdown of dPGC-1. Indeed, using the colorimetric assay of citrate synthase activity described here, we found that knockdown of dRNF34 in Drosophila muscle increased mitochondrial citrate synthase activity, which was reversed by knockdown of dPGC-1. Specifically using the method described here, an initial linear enzymatic rate was established for each assay (Figure 1). The trend lines with their formulas and coefficient of determination (R2) are shown in the graph (Figure 1). The slopes of the trend lines represent the maximal reaction rates, which are equivalents of the maximal citrate synthase activities of the different genotypes. The slopes for the different genotypes are different (Figure 1). The coefficient of determination is closer to 1; the formulas of the trend lines are more reliable. The protein concentration normalized maximal citrate synthase activities of different genotypes were calculated from the Figure 1 data (Figure 2). The maximal citrate synthase activity of fly thoraxes with muscle-specific dRNF34 knockdown increased, which was reversed by muscle-specific dPGC-1 knockdown (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59454/59454fig1a.jpg" /> 
Figure 1: An example of the kinetic curves for the colorimetric assay of citrate synthase activity. The adult fly thorax homogenates of (a) 24B-Gal4&#62;+ (b) 24B-Gal4&#62;dRNF34RNAi(II), and (c) 24B-Gal4&#62;dRNF34RNAi(II),dPGC-1RNAi were subjected to the colorimetric assay of citrate synthase activity. The horizontal axes represent reaction times, and the vertical axes represent OD absorbencies at 412 nm. A linear enzymatic rate was established for each genotype. The trend lines were drawn and the formulas and R2 of the trend lines are shown in the graph. <a href="https://www.jove.com/files/ftp_upload/59454/59454fig1large.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/59454/59454fig2.jpg" /> 
Figure 2: The maximal citrate synthase activities calculated from the kinetic curves and normalized by protein concentration. The maximal citrate synthase activity of fly thoraxes with muscle-specific dRNF34 knockdown increased, which was reversed by muscle-specific dPGC-1 knockdown. All the data are represented as means &#177; SEM (*p &#60; 0.01, by ANOVA test, and Tukey&#39;s test for multiple comparisons, n = 3, 10 thoraxes per replicate). This figure is modified from Wei et al.17. <a href="https://www.jove.com/files/ftp_upload/59454/59454fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

a-colorimetric-assay-citrate-synthase-activity-drosophila

Merck & Co., Inc.

Research

JoVE Journal

Biochemistry

9.9K Views.  Shanghai Jiao Tong University Affiliated Sixth People's Hospital. We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Video: A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

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 ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Author Spotlight: Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...