Name: lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism
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Ethiek Verklaring: Alle protocollen met betrekking tot de behandeling van menselijke monsters volgen de richtlijnen van de Universiteit van Pennsylvania menselijk onderzoek ethische commissie. 1. Bereiding van buffers en 100x Voorraadoplossingen <ol><li> Bereid 1 liter bufferbasis Tyrode. Combineer 8,123 g NaCl, 1,428 g NaHCO3, 0,466 g CaCl2 ∙ 2 H 2 O, 0,224 g KCI en 0,095 g MgCl 2. Pas de totale volume op 1 L met DDH 2 O. Filter ste...

<ol>
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Hier hebben we het nut van geïsoleerde bloedplaatjes weergegeven als een platform voor het bestuderen verstoord mitochondriale metabolisme. Wij hebben in het metabolische aanpassing kenmerk reactie op complex I remming door rotenon. Deze studie werd eerder gemeld bevindingen uitgebreid over de rol van complex I remming door rotenon in cellijnen van menselijke bloedplaatjes. Belangrijk is dat deze is gebleken dat rotenon geremd bloedplaatjes succinyl-CoA vorming stimuleerde een toename van b...

Disfunctionele mitochondriaal metabolisme is betrokken bij een groot aantal ziekten, waaronder neurodegeneratie, kanker en cardiovasculaire aandoeningen 30. Als zodanig is grote inspanning geplaatst op het karakteriseren van metabolische defecten die bijdragen aan pathogenese. Vloeistofchromatografie-tandem massaspectrometrie (LC-MS / MS) wordt beschouwd als de gouden standaard voor de kwantificatie van analyten uit complexe biologische matrices en wordt vaak gebruikt voor metabolismeonderzoek 8. Echter, zoals vaak het geval is bij biomedische studies, bereiken een toegankelijke en goed gedefinieerde model voor menselijke ziekten tegen een uitdaging. Veel studies in dienst getransformeerd cellulaire modellen voor het sonderen van de impact van xenobiotica of genetische afwijkingen op de celstofwisseling 7,9. De metabole herprogrammering die optreedt in kankercellen kan introduceren storende factoren 21 en zijn dus niet ideaal. Deze problemen kunnen worden circumvented met primaire cel modellen, hoewel het verkrijgen van voldoende biomassa voor metabole analyses kan een uitdaging zijn. Bovendien is de impact van grote hoeveelheden antibiotica gebruikt in de cultuur zijn gemarkeerd als potentieel verstorende mitochondriale studies 16. Menselijke bloedplaatjes in de gelegenheid om een primaire cel model met voldoende mitochondriale inhoud voor metabole studies 5,22,27,32 benutten. Ten eerste kan bloedplaatjes gemakkelijk worden verkregen door bloedafnames van individuele donoren, of in grote hoeveelheden uit bloedbanken en daarom een ​​model waarin externe factoren gemakkelijk kan worden gecontroleerd. Ten tweede, als gevolg van hun geringe omvang, bloedplaatjes kan eenvoudig worden geïsoleerd van andere bloedcomponenten met minimale voorbereidende werkzaamheden in zelfs minimaal uitgeruste laboratoria 5. We merken hebben bloedplaatjes niet kernen bevatten en kunnen daarom worden gebruikt om veranderingen onafhankelijk van transcriptionele regulatie te bestuderen metabolisme. Hier laten we zien datnaast relatieve kwantificatie van acyl-coenzym A (CoA) thioesters, kan het geïsoleerde bloedplaatjes systeem worden gebruikt om koolstofmetabolisme onderzoeken. Specifiek melden wij het ​​gebruik van metabolisch labelen met stabiele isotopen (niet-radioactief) gemerkte [13 C 6] glucose en [13 C 16] -palmitate incorporatie van [13 C] -label sonde in de belangrijkste metaboliet acetyl- CoA via glycolyse of vetzuuroxidatie. Dit zorgt voor een krachtige, generaliseerbare en veelzijdig platform vanwege de grote betrokkenheid van acyl-CoA soorten biochemische pathways 13,24 en de traceerbaarheid van dit systeem onderzoeken van andere variabelen, zoals de remming van complex I met rotenon 3,33. In aanvulling op de informatie in de onderstaande protocol, kan een uitgebreide beschrijving van de gebruikte isotoop etikettering en voor de LC-MS-gebaseerde analyses methoden te vinden in Basu en 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.

Om het nut van deze methode hebben we de generaliseerbaarheid van de eerder beschreven compenserende metabolische aanpassing als gevolg van blootstelling aan rotenon gereproduceerd demonstreren. Deze bevinding werd eerder geïdentificeerd in celcultuur modellen en dit onderzoek was erop gericht om te testen of deze metabole verschuiving komt ook voor in bloedplaatjes, die kernloze en niet gevoelig voor dezelfde experimentele artefacten als celcultuur zijn. Dit werk werd uitgevoerd met 6 ...

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

Hier laten we geïsoleerde humane bloedplaatjes kan worden gebruikt als een toegankelijke ex vivo model metabole aanpassingen studeren in reactie op de remmer complex I rotenon. Deze benadering maakt gebruik van isotopische tracering en relatieve kwantificatie door middel van vloeistofchromatografie-massaspectrometrie en kan worden toegepast op een verscheidenheid van studieopzetten.

LC-MS analyse van menselijke bloedplaatjes als een platform voor het bestuderen van mitochondriaal Metabolisme

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

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Unit 1

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SCIENTISTS IN ACTION

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Techniek voor het bestuderen van geleedpotigen en microbiële gemeenschappen binnen 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 ...

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

Voorbijgaande Gene Expression Tobacco gebruik Gibson Vergadering en de 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

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

Gebruik van Chironomidae (Diptera)-Surface Drijvende Pupal Exuviae als Rapid Protocol Bioassessment voor Water Bodies

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

Wijziging en toepassing van een bladblazer-vac voor Field Sampling geleedpotigen

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

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

Thermische Conductometry (TSC) scannen als een algemene methode voor het bestuderen van en het beheren van het gedrag van de fase van geleidende fysieke 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 ...

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

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

Kwantificeren van de Plant oplosbaar eiwit en verteerbare koolhydraten inhoud, met behulp van maïs (Zea mays) als een 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 ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Neurobehavioral Effecten van milieuverontreinigende stoffen op zebravissenlarven bestuderen

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

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

Het effect van bouw- en sloopafval plastic fracties op hout-polymeer composiet eigenschappen

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

Een gnotobiotisch systeem voor het bestuderen van microbioomassemblage in de Phyllosfeer en in plantaardige fermentatie

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

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

Microplot Ontwerp en Plant en Bodem Monster Voorbereiding voor 15Stikstof Analyse

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. ...