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 buffer.</li>
	<li>Prepare 100x stock solutions of glucose and palmitic acid. For 100x glucose stock solutions, dissolve 100 mg glucose or [13C6]-glucose in 1 ml of ddH2O. For 100x palmitate solutions, dissolve 2.56 mg of palmitic acid or 2.72 mg of [13C16]-palmitic acid in 1 ml of ethanol. Filter sterilize 100x stock solutions.</li>
	<li>Prepare Appropriate Enriched Tyrode&#39;s Buffers
	<ol>
		<li>For non-labeling studies, add 1 ml of 100x glucose stock solution and 1 ml of 100x palmitic acid stock solution to 98 ml of base Tyrode&#39;s buffer.</li>
		<li>For glucose labeling studies, add 1 ml of 100x [13C6]-glucose stock solution and 1 ml of 100x palmitic acid stock solution to 98 ml of base Tyrode&#39;s buffer.</li>
		<li>For palmitic acid labeling studies, add 1 ml of 100x glucose stock solution and 1 ml of [13C16]-palmitic acid stock solution to 98 ml of base Tyrode&#39;s buffer.</li>
	</ol>
	</li>
	<li>Prepare Solutions for Rotenone Treatment
	<ol>
		<li>Prepare a 10 mM stock solution of rotenone in dimethyl sulfoxide (DMSO).</li>
		<li>For non-labeling dose-response studies, proceed to 1.4.3. For labeling studies at a single dose, proceed to 1.4.5.</li>
		<li>Perform a serial dilution of the 10 mM rotenone stock in DMSO to obtain solutions with rotenone concentrations ranging from 1 nM to 100 &#181;M. These are 100x stocks.</li>
		<li>Add 50 &#181;l of each stock to 5 ml of base Tyrode&#39;s buffer + glucose + palmitic acid from 1.3.1 to prepare solutions with rotenone concentrations ranging from 10 pM to 1 &#181;M. Alternatively, add 50 &#181;l of DMSO to serve as a vehicle control. Proceed to 2.1.</li>
		<li>Dilute the 10 mM stock solution 1,000-fold in DMSO to obtain a 10 &#181;M working solution.</li>
		<li>Add 50 &#181;l of this stock to 5 ml each of base Tyrode&#39;s buffer + [13C6]-glucose + palmitic acid from 1.3.2 and base Tyrode&#39;s buffer + glucose + [13C16]-palmitic acid from 1.3.3. The final concentration is 100 nM rotenone. Alternatively, add 50 &#181;l of DMSO to each buffer to serve as vehicle controls.</li>
	</ol>
	</li>
</ol>
2. Platelet Isolation
Note: This method is amenable to platelets derived from either whole blood or from platelet bags. The example data contained herein was prepared using platelets derived from platelet bags. Please see Basu et al.5 for more details regarding using platelets isolated from whole blood.
<ol>
	<li>Platelet Isolation from Whole Blood 
	NOTE: If isolating platelets from a bag of platelets, proceed to 2.2.1.
	<ol>
		<li>Centrifuge whole blood at 175 x g for 15 min with no brakes.</li>
		<li>Transfer 1 ml of the upper platelet-rich plasma (PRP) layer to a 1.5 ml microcentrifuge tube.</li>
		<li>Centrifuge the PRP at 400 x g for 5 min.</li>
		<li>Aspirate supernatant and proceed to step 3.1 or 3.2.</li>
	</ol>
	</li>
	<li>Platelet Isolation from a Platelet Bag
	<ol>
		<li>Transfer 1 ml of the platelet suspension to a 1.5 ml microcentrifuge tube.</li>
		<li>Centrifuge the suspension at 400 x g for 5 min.</li>
		<li>Aspirate supernatant and proceed to step 3.1. or 3.2.</li>
	</ol>
	</li>
</ol>
3. Performing an Experiment
<ol>
	<li>To determine the effect of xenobiotic treatment (e.g., rotenone) on the levels of a metabolite of interest (e.g., acetyl-CoA), proceed to 3.1.1. To determine the effect of a xenobiotic (e.g., rotenone) on the incorporation of a metabolic precursor into a downstream metabolite of interest, proceed to 3.2.1.
	<ol>
		<li>Using no fewer than three biological replicates for each condition, resuspend platelet pellet from step 2.1.4 or 2.2.3 in 1 ml of each solution from step 1.4.4.</li>
		<li>Incubate resuspended platelets in a water-jacketed CO2 incubator set to 95% humidity, 5% CO2 and 37 &#176;C for 1 hr. Proceed to step 4.1 
		Note: Here we describe a dose response experiment. Alternatively, a time course experiment could be done in which the dose was fixed and the length of incubation was varied instead.</li>
	</ol>
	</li>
	<li>To determine the effect of xenobiotic treatment on incorporation of a metabolic precursor into a downstream metabolite of interest.
	<ol>
		<li>Using no fewer than three biological replicates for each condition, resuspend platelet pellet from step 2.1.4 or 2.2.3 in 1ml of each formulation of labeled Tyrode&#39;s buffer from step 1.4.6.</li>
		<li>Incubate resuspended platelets in a water-jacketed CO2 incubator set to 95% humidity, 5% CO2 and 37 &#176;C for 1 hr. Proceed to step 4.1.</li>
	</ol>
	</li>
</ol>
4. Quenching and CoA Extraction
<ol>
	<li>Pellet platelets by centrifuging at 3,000 x g for 3 min.</li>
	<li>Aspirate supernatant.</li>
	<li>Resuspend platelets in 750 &#181;l ice cold 10% trichloroacetic acid (w/v).</li>
	<li>Add appropriate stable isotope-labeled internal standard. For example, if the goal is to compare the levels of CoA species across samples, use 100 &#181;l of a mixture of stable-isotope labeled CoA thioesters obtained from yeast as described previously (SILEC technique) 29.</li>
	<li>Pulse-sonicate each sample 30 times with 0.5 sec pulses.</li>
	<li>Centrifuge samples at 16,000 x g for 10 min at 4 &#176;C.</li>
	<li>Affix C18 solid-phase extraction (SPE) columns to vacuum manifold.</li>
	<li>Condition the columns with 1 ml methanol.</li>
	<li>Equilibrate the columns with 1 ml ddH2O.</li>
	<li>Run sample-derived supernatant (approximately 850 &#181;l in this case) through the columns.</li>
	<li>Wash the columns with 1 ml water.</li>
	<li>Load 10 ml glass centrifuge tubes into the vacuum manifold to collect elution fraction.</li>
	<li>Elute the columns with 1 ml of 25 mM ammonium acetate in methanol.</li>
	<li>Dry eluate under nitrogen gas.</li>
	<li>Resuspend dried residues in 50 &#181;l 5% (w/v) 5-sulfosalicylic acid and transfer into HPLC vials.</li>
</ol>
5. HPLC Setup
<ol>
	<li>Using the control software for the HPLC system, create a method with HPLC conditions optimized for the target analytes and utilized column, as listed in Table 1. 
	Note: The column temperature used for the generation of this data was 25 &#176;C but the specific parameters will vary by instrument and with respect to the compounds of interest. For acyl-CoA quantitation, multiple methods of LC-MS analysis have been reported, and validated for different analytes in different LC conditions 14,19,20.</li>
	<li>Allow the HPLC to Adequately Equilibrate. 
	Note: This should include both priming of solvents before attaching a column and equilibration of the column at the starting solvent conditions for the method. The equilibration volume should follow the manufacturer&#39;s directions, and resulting effluent should be diverted to waste. 
	Note: Consult Basu and Blair 5 for more details regarding the HPLC settings.</li>
</ol>
6. Mass Spectrometer Setup
<ol>
	<li>Using the control software for the mass spectrometer, create a targeted method for detection of ionized forms of the compounds of interest. Parameters are presented in Table 2. 
	Note: Stable isotope analogues of these compounds should be used to determine whether a positive or negative mode gives superior sensitivity and to optimize the source and optics condition for their detection. The total detection time of the mass spectrometer method should correspond to the time component of the associated HPLC method. 
	Note: As previously mentioned, multiple methods of acyl-CoA analysis have been reported, including positive 14 and negative ionization 15 modes and use of a high resolution mass spectrometer 17 rather than a triple quadrupole instrument 28.</li>
	<li>Clean and calibrate the instrument, establish a stable spray into the spectrometer, and allow time for the calibration solution to adequately dissipate from the source and optics according to manufacturer&#39;s instructions.</li>
	<li>Set up a sequence for all the samples with the mass spectrometer control software. Include the name or numerical identifiers and indicate the appropriate injection volume and instrument method. Run a blank before the first sample, and run other blanks and washes between samples as needed to mitigate carryover or matrix effects. 
	Note: A processing method for peak integration of selected analytes&#39; liquid chromatograms can often be set within this sequence or subsequently applied in data processing.</li>
	<li>Begin the sequence and monitor the mass spectrometer and HPLC system periodically for problems including pressure fluctuations or system failures and loss of signal intensity during the run. 
	Note: Severe problems or persistent run failures may require stopping the run and purging and re-equilibrating the HPLC system as well as cleaning and calibrating the mass spectrometer. Consult Basu and Blair 5 for more details regarding the MS settings and data processing.</li>
</ol>

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The authors declare no competing financial interests.

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;HB-CoA and had no effect on platelet levels of acetyl-CoA.
Great care must be taken to ensure the validity and reproducibility of any analysis of this type. If platelets are to be isolated from whole blood, the isolation should proceed exactly as described above in order to prevent lymphocyte contamination and platelet activation, with special attention devoted to leaving the buffy coat containing the lymphocytes undisturbed. In order for dose response curves to be meaningful, serial dilutions must be performed precisely, with mixing to homogeneity at each step. After platelet resuspension, incubation conditions may be modified from those detailed above, but they must be rigorously standardized across treatment groups to allow for meaningful comparisons to be made. Perhaps the most important step in any quantitative LC/MS-based analysis is the use of stable-isotope labeled internal standards.
The use of internal standards is critical in this setting because it protects against potentially confounding &#34;batch effects&#34; such as variable extraction efficiencies and analyte stability across samples. Addition of the internal standard, (again, mixing to homogeneity) as early as possible in an experimental protocol reduces the potential for artifacts. Finally, as in any experiment, the use of multiple replicates for each experimental condition is essential, and if feasible, power calculations based on pilot experiments may be useful.
Platelets can be rapidly isolated from individual or pooled sources 5,27,32, making this approach amenable to a wide range of applications. It is important to note that the ability to perform studies involving both relative quantification and isotopic labeling allows for a more comprehensive characterization of a metabolic system. Currently, this demands two separate assays, which increases the demand for biomass required for the experiment. For this reason, platelets, which can be readily obtained in large numbers, are more efficient than other cell culture models.
However, despite the numerous advantages conferred by the use of platelets, there are drawbacks. First, care must be taken in the selection of human donors because there are many disorders that affect platelet physiology 25. In addition, platelet activation during the course of sample preparation and analysis is a potential confounding variable in any platelet-based assay. For these reasons, concomitant assessment of markers of platelet activation by ELISA 26, flow cytometry 1, or assays such as the light transmission aggregometry (LTA) assay 34 could prove prudent.
Although the present study has focused on the analysis of acyl-CoA thioesters, this approach can be readily expanded to include a wider range of analytes. Untargeted metabolomics approaches are providing mechanistic insight into an array of disease states 23. Applying such untargeted approaches to platelet models will likely provide additional insight into the underlying biochemical mechanisms involved in dysregulated cellular metabolism.
This approach can be readily expanded to include investigations of other environmental chemicals and drugs known to interfere with mitochondrial metabolism 6,12,18,31 as well as a system to test the effects of xenobiotics as potential modulators of CoA metabolism 10. In addition, the general approach can be employed to study metabolic defects in genetic diseases such as Friedreich&#39;s ataxia 5,32. Moreover, any of the aforementioned experimental designs could be coupled with a functional respiration assay in order to more robustly characterize the state of mitochondria in each context 2. Thus, coupling stable isotopes and LC-MS analysis with metabolic studies in isolated platelets is likely to afford novel opportunities to further characterize aberrant mitochondrial metabolism.

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><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>746398</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt; * H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>208337</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;-Glucose</td><td>Sigma-Aldrich</td><td>389374</td><td></td></tr><tr><td>Palmitic acid</td><td>Cayman</td><td>10006627</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;16&lt;/sub&gt;-Palmitic Acid</td><td>Sigma-Aldrich</td><td>605573</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Trichloro Acetic Acid</td><td>Sigma-Aldrich</td><td>T6399</td><td></td></tr><tr><td>5-Sulfosalicylic Acid</td><td>Sigma-Aldrich</td><td>390275</td><td></td></tr><tr><td>Acetonitirle</td><td>Fischer Scientific</td><td>A996-4</td><td>(optima)</td></tr><tr><td>Water (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fischer Scientific</td><td>W7-4</td><td>(optima)</td></tr><tr><td>Formic acid</td><td>Fischer Scientific</td><td>85171</td><td>(optima)</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich</td><td>472301</td><td></td></tr><tr><td>Ethanol</td><td>Fischer Scientific</td><td>04-355-222</td><td></td></tr><tr><td>Methanol</td><td>Fischer Scientific</td><td>A454-4</td><td>(optima)</td></tr><tr><td>Ammonium Acetate</td><td>Fischer Scientific</td><td>A639-500</td><td></td></tr><tr><td>2 ml Eppendorf Tubes</td><td>BioExpress</td><td>C-3229-1</td><td></td></tr><tr><td>LC vials (plastic)</td><td>Waters</td><td>186002640</td><td></td></tr><tr><td>10 ml Glass Centrifuge Tubes</td><td>Kimble Chase</td><td>73785-10</td><td></td></tr><tr><td>Oasis Solid Phase Extraxtion (SPE) Columns</td><td>Waters</td><td>WAT094225</td><td></td></tr><tr><td>Pastuer Pipets</td><td>Fischer Scientific</td><td>13-678-200</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Water-Jacketed Incubator</td><td>Nuaire</td><td>AutoFlow NU-8500</td><td></td></tr><tr><td>Triple Quadropole Mass Spectrometer</td><td>Thermo Scientific</td><td>Finnigan TSQ Quantum</td><td></td></tr><tr><td>HPLC</td><td>Thermo Scientific</td><td>Dionex Ultimate 3000</td><td></td></tr><tr><td>Source</td><td>Thermo Scientific</td><td>HESI II</td><td></td></tr><tr><td>HPLC Column</td><td>Phenomenex</td><td>Luna C18</td><td>3 &amp;mu;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 Center, which had been deemed too old for human infusion, although they retained their metabolic activity. The isolation was performed as shown in Figure 1. The total workflow can be scaled easily from a limited number of clinical samples or experimental conditions to 96-well plate based higher throughput with the availability of platelets that are no longer useful for infusion into patients.
Our group has previously reported changes in relative levels of acyl-CoA thioesters in response to rotenone in cell lines that are hypothesized to result from the inhibition of complex I (Figure 2). Specifically we have observed in SH-SY5Y cells a dose dependent decrease in succinyl-CoA (IC50 = 25 nM) with a simultaneous increase in &#946;-hydroxybutyryl (HB)-CoA (ED50 = 75 nM), while acetyl-CoA levels were unchanged 1. Our analysis of human platelets treated with rotenone revealed highly consistent results (Figure 3). This provides a unique set of experimental data in human platelets pointing to the mitochondrial dependence of the response to rotenone. This response cannot be mediated through conventional transcriptional mechanisms because platelets lack nuclei.
Relative quantification provides one dimension of metabolic information, but isotopic labeling provides invaluable complementary insight into the activity of specific metabolic pathways. This is critical in metabolic studies because multiple pathways may converge through a single intermediate. To demonstrate this point, platelets were treated with either 100 nM rotenone or DMSO in the presence of either [13C6]-glucose or [13C16]-palmitate for 1 hr. Co-elution of isotopologues of acyl-CoAs from the LC column allows for definitive determination of isotopic composition of analytes (Figure 4). Following correction for the natural abundance of heavy isotopes as described 11 this approach showed a significant decrease in glycolytic production of acetyl-CoA in the platelets while palmitate incorporation was increased significantly (Figure 5). These findings are similar to those observed previously in the SH-SY5Y cell culture model 33 and so provide additional evidence that this pathway could be important in vivo.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>0</td>
			<td>0</td>
			<td>1.5</td>
			<td>5</td>
			<td>12</td>
			<td>17</td>
			<td>18</td>
			<td>23</td>
			<td>25</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Total Flow (&#956;l/min)</td>
			<td>200</td>
			<td>200</td>
			<td>200</td>
			<td>200</td>
			<td>200</td>
			<td>250</td>
			<td>200</td>
			<td>200</td>
			<td>200</td>
			<td>200</td>
		</tr>
		<tr>
			<td>% A</td>
			<td>98</td>
			<td>98</td>
			<td>98</td>
			<td>80</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>98</td>
			<td>98</td>
		</tr>
		<tr>
			<td>% B</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
			<td>20</td>
			<td>100</td>
			<td>100</td>
			<td>0</td>
			<td>0</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>% C</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>100</td>
			<td>100</td>
			<td>0</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Liquid Chromatography Gradient. Solvent A: 5 mM Ammonium Acetate in Water, Solvent B: 5 mM Ammonium Acetate in 95:5 ACN/Water Solvent C: 80:20 ACN/Water 0.1% Formic Acid
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Spray Voltage (V)</td>
			<td>4,000</td>
		</tr>
		<tr>
			<td>Vaporizer Temperature (&#176;C)</td>
			<td>400</td>
		</tr>
		<tr>
			<td>Sheath Gas Pressure</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Auxiliary Gas Pressure</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Capillary Temperature&#160; (&#176;C)</td>
			<td>350</td>
		</tr>
		<tr>
			<td>Tube Lens Offset (V)</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
Table 2. Mass Spectrometer Parameters.
<img alt="Figure 1" src="/files/ftp_upload/53941/53941fig1.jpg" /> 
Figure 1. Generalized Workflow for Sample Preparation and Analysis. This schematic depicts the workflow for platelet isolation and treatment for metabolic studies by LC-MS/MS analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53941/53941fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53941/53941fig2.jpg" /> 
Figure 2. Metabolic Scheme of Glucose and Lipid Metabolism and the Electron Transport Chain. Acetyl-CoA can be synthesized from either glucose or palmitate. Rotenone inhibits the proper shuttling of electrons by mitochondrial complex I. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53941/53941fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53941/53941fig3.jpg" /> 
Figure 3. Quantitation of CoA-thioesters in Response to Rotenone. Rotenone treatment does not affect acetyl-CoA levels but induces a dose-dependent decrease in succinyl-CoA (IC50 = 4.1 nM) with a concomitant increase in &#946;HB-CoA (ED50 = 4.2 nM). Error bars represent standard error of the mean (SEM); n = 4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53941/53941fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53941/53941fig4.jpg" /> 
Figure 4. Representative Chromatograms Showing Isotopic Labeling from Palmitate into Acetyl-CoA. Rotenone treatment increases the incorporation of palmitate into acetyl-CoA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53941/53941fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53941/53941fig5.jpg" /> 
Figure 5. Relative Incorporation of [13C6]-glucose or [13C16]-palmitate into Acetyl-CoA. Rotenone treatment decreases glucose incorporation into acetyl-CoA and increases palmitate incorporation. Error bars represent SEM; n = 4. Asterisks denote p &#60;0.05 (Student&#39;s unpaired t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53941/53941fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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11.2K Views.  University of Pennsylvania.  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. 

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

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Biochemistry

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Cancer Research

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Immunology and Infection

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Neuroscience

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...

Biology

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these ...

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

Isolating Quiescent Platelets for Metabolic Research

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%&#8211;40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.

Platelet metabolism is of interest, particularly as it relates to the role of platelet hyper- and hypoactivity in bleeding and thrombotic disorders. Isolating platelets from plasma is necessary for some metabolic assays; presented here is a method for isolating of intracellular metabolites from washed platelets.

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated ...

Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Source: Maxwell, J.T., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
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LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

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