We acknowledge the support of NIH grants P30ES013508 and T32ES019851.

Ethics Statement: All protocols concerning the treatment of human samples follow the guidelines of The University of Pennsylvania&#39;s human research ethics committee.
1. Preparation of Buffers and 100x Stock Solutions
<ol>
	<li>Prepare 1 L of base Tyrode&#39;s buffer. Combine 8.123 g NaCl, 1.428 g NaHCO3, 0.466 g CaCl2&#8729;2 H2O, 0.224 g KCl, and 0.095 g MgCl2. Adjust total volume to 1 L with ddH2O. Filter sterilize base Tyrode&#39;s...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelets+as+a+platform+to+monitor+metabolic+biomarkers+using+stable+isotopes+and+LC-MS.">Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.</a> Bioanalysis. 5 (24), 3009-3021 (2013).</li><li>Berson, A., 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=Mechanisms+for+experimental+buprenorphine+hepatotoxicity:+major+role+of+mitochondrial+dysfunction+versus+metabolic+activation.">Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.</a> J. Hepatol. 34 (2), 261-269 (2001).</li><li>Castell, J. V., Jover, R., Martinez-Jimenez, C. P., Gomez-Lechon, M. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Links+between+metabolism+and+cancer.">Links between metabolism and cancer.</a> Genes Dev. 26 (9), 877-890 (2012).</li><li>Darnell, M., Weidolf, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+xenobiotic+carboxylic+acids:+focus+on+coenzyme+A+conjugation,+reactivity,+and+interference+with+lipid+metabolism.">Metabolism of xenobiotic carboxylic acids: focus on coenzyme A conjugation, reactivity, and interference with lipid metabolism.</a> Chem. Res. Toxicol. 26 (8), 1139-1155 (2013).</li><li>Des Rosiers, C., Fernandez, C. 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Lipid Res. 49 (5), 1113-1125 (2008).</li><li>Ikegawa, 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=Characterization+of+cholyl-adenylate+in+rat+liver+microsomes+by+liquid+chromatography/electrospray+ionization-mass+spectrometry.">Characterization of cholyl-adenylate in rat liver microsomes by liquid chromatography/electrospray ionization-mass spectrometry.</a> Anal. Biochem. 266 (1), 125-132 (1999).</li><li>Kalghatgi, 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=Bactericidal+antibiotics+induce+mitochondrial+dysfunction+and+oxidative+damage+in+Mammalian+cells.">Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in Mammalian cells.</a> Sci. Transl. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC/MS/MS+method+for+quantitative+determination+of+long-chain+fatty+acyl-CoAs.">LC/MS/MS method for quantitative determination of long-chain fatty acyl-CoAs.</a> Anal. Chem. 77 (9), 2889-2894 (2005).</li><li>Mauriala, T., Herzig, K. H., Heinonen, M., Idziak, J., Auriola, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+long-chain+fatty+acid+acyl-coenzyme+A+compounds+using+liquid+chromatography-electrospray+ionization+tandem+mass+spectrometry.">Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry.</a> J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 808 (2), 263-268 (2004).</li><li>Murphy, T. A., Dang, C. V., Young, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopically+nonstationary+13C+flux+analysis+of+Myc-induced+metabolic+reprogramming+in+B-cells.">Isotopically nonstationary 13C flux analysis of Myc-induced metabolic reprogramming in B-cells.</a> Metab Eng. 15, 206-217 (2013).</li><li>Paglia, 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=Metabolomic+analysis+of+platelets+during+storage:+a+comparison+between+apheresis-+and+buffy+coat-derived+platelet+concentrates.">Metabolomic analysis of platelets during storage: a comparison between apheresis- and buffy coat-derived platelet concentrates.</a> Transfusion. 55 (2), 301-313 (2015).</li><li>Patti, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+strategies+for+untargeted+metabolomics.">Separation strategies for untargeted metabolomics.</a> J. Sep. Sci. 34 (24), 3460-3469 (2011).</li><li>Robishaw, J. D., Neely, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coenzyme+A+metabolism.">Coenzyme A metabolism.</a> Am. J. Physiol. 248 (1 Pt 1), E1-E9 (1985).</li><li>Rodgers, 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=Overview+of+platelet+physiology+and+laboratory+evaluation+of+platelet+function.">Overview of platelet physiology and laboratory evaluation of platelet function.</a> Clin. Obstet. Gynecol. 42 (2), 349-359 (1999).</li><li>Salles, I., 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=Development+of+a+high-throughput+ELISA+assay+for+platelet+function+testing+using+platelet-rich+plasma+or+whole+blood.">Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.</a> Thromb. Haemost. 104 (2), 392-401 (2010).</li><li>Snyder, N. W., Basu, S. S., Worth, A. J., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+propionic+acid+to+a+novel+acyl-coenzyme+A+thioester+by+mammalian+cell+lines+and+platelets.">Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets.</a> J. Lipid Res. 56 (1), 142-150 (2015).</li><li>Snyder, N. W., Basu, S. 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Biochem. 474, 59-65 (2015).</li><li>Wallace, 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=Mitochondrial+diseases+in+man+and+mouse.">Mitochondrial diseases in man and mouse.</a> Science. 283 (5407), 1482-1488 (1999).</li><li>Wojtovich, A. P., Brookes, P. 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+complex+II+inhibitor+atpenin+A5+protects+against+cardiac+ischemia-reperfusion+injury+via+activation+of+mitochondrial+KATP+channels.">The complex II inhibitor atpenin A5 protects against cardiac ischemia-reperfusion injury via activation of mitochondrial KATP channels.</a> Basic Res. Cardiol. 104 (2), 121-129 (2009).</li><li>Worth, A. 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=Stable+isotopes+and+LC-MS+for+monitoring+metabolic+disturbances+in+Friedreich's+ataxia+platelets.">Stable isotopes and LC-MS for monitoring metabolic disturbances in Friedreich's ataxia platelets.</a> Bioanalysis. 7 (15), 1843-1855 (2015).</li><li>Worth, A. J., Basu, S. S., Snyder, N. W., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neuronal+cell+mitochondrial+complex+I+with+rotenone+increases+lipid+beta-oxidation,+supporting+acetyl-coenzyme+A+levels.">Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid beta-oxidation, supporting acetyl-coenzyme A levels.</a> J. Biol. Chem. 289 (39), 26895-26903 (2014).</li><li>Zhou, L., Schmaier, A. 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Here we have shown the utility of isolated platelets as a platform for studying perturbed mitochondrial metabolism. Specifically, we have characterized metabolic adaptation in response to complex I inhibition by rotenone.
The present study has extended previously reported findings on the role of complex I inhibition by rotenone in cell lines to human platelets. Importantly, this has revealed that rotenone also inhibited platelet succinyl-CoA formation, stimulated an increase in platelet &#946;...

Dysfunctional mitochondrial metabolism has been implicated in a wide range of diseases including neurodegeneration, cancer, and cardiovascular disease 30. As such, great effort has been placed on characterizing metabolic defects that contribute to disease pathogenesis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the gold standard for quantification of analytes from complex biological matrices and is often employed for metabolic studies 8. However, as is often the case with biomedical studies, attaining an accessible and well-defined model relevant to human disease is a challenge.
Many studies employ transformed cellular models for probing the impact of xenobiotics or genetic abnormalities on cellular metabolism 7,9. The metabolic reprogramming that occurs in cancer cells can introduce confounding factors 21 and are therefore not ideal. These issues can be circumvented with primary cell models, although obtaining sufficient biomass for metabolic analyses can be challenging. Furthermore, the impact of high amounts of antibiotics used in culture has been highlighted as potentially confounding mitochondrial studies 16.
Human platelets afford the opportunity to utilize a primary cell model with sufficient mitochondrial content for metabolic studies 5,22,27,32. First, platelets can be easily acquired, through blood draws from individual donors, or in large volumes from blood banks, and therefore provide a model in which external factors can be readily controlled. Secondly, due to their small size, platelets can be easily isolated from other blood components with minimal preparatory work in even minimally equipped laboratories 5. Of note, platelets do not contain nuclei and can therefore be used to study alterations to metabolism independently of transcriptional regulation. Here we show that in addition to relative quantification of acyl-coenzyme A (CoA) thioesters, the isolated platelet system can be used to examine carbon metabolism. Specifically, we report the use of metabolic labeling with stable isotope (non-radioactive) labeled [13C6]-glucose and [13C16]-palmitate to probe incorporation of the [13C]-label into the important metabolite acetyl-CoA via glycolysis or fatty acid oxidation. This provides a powerful, generalizable, and versatile platform due to the extensive involvement of acyl-CoA species in biochemical pathways 13,24 and the tractability of this system to testing other variables, such as inhibition of complex I with rotenone 3,33. In addition to information provided in the Protocol below, an extensive description of the methods used for isotope labeling and for the LC-MS-based analyses can be found in Basu and Blair 4.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Reagent</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Chloride (NaCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">746398</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Bicarbonate (NaHCO3)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">S5761</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Calcium Chloride Dihydrate (CaCl2 * H2O)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">223506</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Potassium Chloride (KCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">P9541</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">208337</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">G8270</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C6-Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td>389374</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Palmitic acid</td>
			<td style="width:92px;">Cayman</td>
			<td style="width:68px;">10006627</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C16-Palmitic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">605573</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Rotenone</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">R8875</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Trichloro Acetic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">T6399</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">5-Sulfosalicylic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">390275</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Acetonitirle</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A996-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Water (H2O)</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">W7-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Formic acid</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">85171</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Dimethyl Sulfoxide</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">472301</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ethanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">04-355-222</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Methanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A454-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ammonium Acetate</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A639-500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">2 mL Eppendorf Tubes</td>
			<td style="width:92px;">BioExpress</td>
			<td style="width:68px;">C-3229-1</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">LC vials (plastic)</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">186002640</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">10 mL Glass Centrifuge Tubes</td>
			<td style="width:92px;">Kimble Chase</td>
			<td style="width:68px;">73785-10</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Oasis Solid Phase Extraxtion (SPE) Columns</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">WAT094225</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Pastuer Pipets</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">13-678-200</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Name</td>
			<td style="width:92px;">Company</td>
			<td style="width:68px;">Catalog Number</td>
			<td style="width:85px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Equipment</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:161px;">CO2 Water-Jacketed Incubator</td>
			<td style="width:92px;">Nuaire</td>
			<td style="width:68px;">AutoFlow NU-8500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">Triple Quadropole Mass Spectrometer</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Finnigan TSQ Quantum</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">HPLC</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Dionex Ultimate 3000</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Source</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">HESI II</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:161px;">HPLC Column</td>
			<td style="width:92px;">Phenomenex</td>
			<td style="width:68px;">Luna C18</td>
			<td style="width:85px;">3 &#956;m particle size, 200 mm x 2 mm</td>
		</tr>
	</tbody>
</table>

lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

To demonstrate the utility of this methodology we have reproduced the generalizability of previously described compensatory metabolic adaptation resulting from exposure to rotenone. This finding was previously identified in cell culture models and this investigation was aimed to test if this metabolic shift also occurs in platelets, which are anuclear and not prone to the same experimental artifacts as cell culture. This work was performed with 6-day-old platelets from the Penn Trauma Cen...

Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

The overall goal of this procedure is to determine how either a xenobiotic treatment or a disease state alters cellular metabolism. This method has the potential to reveal how a disease state or an applied toxin affects cellular metabolism. And once this is known, our method has the ability to reveal whether some kind of intervention can correct the defect.The main advantage of this technique is it doesn't rely on transformed cell lines, so it's more likely to provide answers that are directly relevant to human health. To begin this procedure prepare a 10 millimolar stock solution of rotenone in dimethyl sulfoxide. Perform a serial dilution of the 10 millimolar rotenone stock solution to obtain solutions with rotenone concentrations ranging from one nanomolar to 100 micromolar.Next, add 50 microliters of each stock solution to five milliliters of a previously-prepared enriched Tyrode's buffer solution to prepare solutions with rotenone concentrations ranging from 10 picomolar to one micromolar. Add 50 microliters of DMSO to one of the buffer solution samples to serve as a vehicle control. To isolate platelets from whole blood, centrifuge whole blood samples at 175 x g for 15 minutes with no breaks.When finished, transfer one milliliter of the upper platelet-rich plasma layer to a 1.5 milliliter microcentrifuge tube. When transferring the platelet-rich plasma, be careful not to disturb the buffy coat so the platelet pellet is not contaminated by lymphocytes. Centrifuge the platelet-rich plasma layer at 400 x g for five minutes.Then, aspirate the supernatent. Using no fewer than three biological replicates for each condition, resuspend the platelet pellet in one milliliter of each previously-prepared rotenone solution. Following this, incubate the resuspended platelets in a water-jacketed carbon dioxide incubator set to 95%humidity and 5%carbon dioxide at 37 degrees Celsius for one hour.At this point, pellet the platelets by centrifuging the samples at 3000 x g for three minutes. After aspirating the supernatent, add the appropriate stable isotope-labelled internal standard. Then, resuspend the platelets in 750 microliters of ice-cold 10%trichloroacetic acid.Pulse sonicate each sample 30 times with 0.5 second pulses. Following centrifugation, affix C18 solid phase extraction columns to a vacuum manifold. Condition the columns with one milliliter of methanol.Then, equilibrate the columns with one milliliter of double-distilled water. Run the sample-derived supernatent through the columns. When finished, wash the columns with one milliliter of water.Next, load 10 milliliter glass centrifuge tubes into the vacuum manifold to collect the elution fractions. Elute the columns with one milliliter of 25 millimolar ammonium acetate and methanol. After drying the eluate under nitrogen gas, resuspend the dried residues in 50 microliters of 5%5-sulfosalicyclic acid and transfer the samples to HPLC files for analysis.As previously reported in SH-SY5Y cells, changes in relative levels of aceyl CoA thioesters in response to rotenone are hypothesized to result from the inhibition of complex one. Specifically, there is a dose-dependent decrease in succinyl CoA with a simultaneous increase in beta-Hydroxybutyryl-CoA, while acetyl CoA levels were unchanged. The analysis of human platelets treated with rotenone reveals highly consistent results and provides a unique set of experimental data.It is important to note that this observation underscores the mitochondrial dependence of the response to rotenone because platelets lack nuclei. Following this procedure, other methods like metabolic tracing can be performed in order to answer additional questions, such as:How does this treatment or disease state affect substrate utilization? If we identify changes in the levels of some metabolite, why is it changing?Which steps in the pathway are affected, and in which way? After watching this video, you should have a good understanding of how to isolate platelets from whole blood, perform the controlled ex-vivo challenge and extract the aceyl CoA thioesters for LCMS anaylsis.

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SCIENTISTS IN ACTION

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to follow the dynamically changing state of the ionogels, and to deliver more information and details about the subtle change of conductive properties with an increase or decrease in the temperature. Moreover, the method allows the performance of long term (i.e. days, weeks) measurements at a constant temperature to investigate the stability and durability of the system and the aging effects. The main advantage of the TSC method over classical conductometry is the ability to perform measurements during the gelation process, which was impossible with the classical method due to temperature stabilization, which usually takes a long time before the individual measurement. It is a well-known fact that to obtain the physical gel phase, the cooling stage must be fast; moreover, depending on the cooling rate, different microstructures can be achieved. The TSC method can be performed with any cooling/heating rate that can be assured by the external temperature system. In our case, we can achieve linear temperature change rates between 0.1 and approximately 10 &#176;C/min. The thermal scanning conductometry is designed to work in cycles, continuously changing between heating and cooling stages. Such an approach allows study of the reproducibility of the thermally reversible gel-sol phase transition. Moreover, it allows the performance of different experimental protocols on the same sample, which can be refreshed to initial state (if necessary) without removal from the measuring cell. Therefore, the measurements can be performed faster, in a more efficient way, and with much higher reproducibility and accuracy. Additionally, the TSC method can be also used as a tool to manufacture the ionogels with targeted properties, like microstructure, with an instant characterization of conductive properties.

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The kinetics of the cooling process defines the properties of ionic gels based on low molecular weight gelators. This manuscript describes the usage of thermal scanning conductometry (TSC), which obtains full control over the gelation process, along with in situ measurements of the samples' temperature and conductivity.

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for plastic materials to be re-utilized, they must be identified and separated according to their polymer composition. In this study, the identification of these materials was performed using near-infrared spectroscopy (NIR), which identified material based on their physical-chemical properties. Advantages of the NIR method are a low environmental impact and rapid measurement (within a few seconds) in the spectral range of 1600-2400 nm without special sample preparation. Limitations include its inability to analyze dark materials. The identified polymers were utilized as a component for wood-polymer composite (WPC) that consists of a polymer matrix, low cost fillers, and additives. The components were first compounded with an agglomeration apparatus, followed by production by extrusion. In the agglomeration process, the aim was to compound all materials to produce uniformly distributed and granulated materials as pellets. During the agglomeration process, the polymer (matrix) was melted and fillers and other additives were then mixed into the melted polymer, being ready for the extrusion process. In the extrusion method, heat and shear forces were applied to a material within the barrel of a conical counter-rotating twin-screw type extruder, which reduces the risk of burning the materials and lower shear mixing. The heated and sheared mixture was then conveyed through a die to give the product the desired shape. The above-described protocol proved the potential for re-utilization of CDW materials. Functional properties must be verified according to the standardized tests, such as flexural, tensile, and impact strength tests for the material.

Secondary material streams have been shown to include potential raw materials for production. Presented here is a protocol in which CDW-plastic waste as a raw material is identified, followed by various processing steps (agglomeration, extrusion). As a result, a composite material was produced, and mechanical properties were analyzed.

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for ...

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...