Name: measurement of oxygen consumption rate in acute mouse brain slices
Uploaded: 2025-04-28T12:01:38.000+00:00
Duration: 1 min 30 s
Description: Source: Zhi, L. et al., Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice. J. Vis. Exp. (2022).This video demonstrates how ...

measurement of oxygen consumption rate in acute mouse brain slices

Measurement of Oxygen Consumption Rate in Acute Mouse Brain Slices

Suction one piece of punched brain tissue, and carefully place the tissue onto the mesh side of the capture screen. A circular piece of plastic with the mesh attached to one side. Using a paper tissue, gently dry the capture screen and remove the moisture, which allows the tissue to become sticky and attached to the center of the mesh.Next, hold the capture screen, slice-side down with tweezers, and place it into one of the wells of the incubating islet plate. Then, incubate the islet plate at 37 degrees Celsius in an incubator for at least 30 minutes, to allow temperature and pH equilibration before running the assay. Dilute the desired compounds and modified artificial cerebrospinal fluid without BSA to the final stock concentration of the working concentration for ports A to D, respectively.Gently preload 75 microliters of the diluted compounds into the appropriate injection ports of the sensor cartridge by placing the tips halfway into the injection ports at a 45-degree angle, with the tip against the wall of the injection port, as complete insertion may cause compound leakage through the port. Afterward, withdraw the tips from the ports carefully without creating air bubbles, and do not tap any portion of the cartridge to avoid alleviating air bubbles. Then, visually inspect the injection ports for even loading, ensuring all liquid is in the port and no residual drops are present on top of the cartridge.After placing the sensor cartridge onto the utility plate, put it into an incubator for 30 minutes to allow it to heat up to 37 degrees Celsius while handling carefully by only holding onto the utility plate and moving as little as possible. First, load the assay template in the software, and then press the green Start button. Then load the sensor cartridge on the utility plate into the instrument tray, ensuring that the plate sits correctly and is flat, loading the drug-filled sensor cartridge into the analyzer for calibration.Afterward, follow the instructions on the screen in order to calibrate and equilibrate the sensors. Once the equilibration step is done, remove the calibration plate, and replace it with the islet plate containing the mesh and tissue slices. Then measure the oxygen consumption rate in each well of the plate, using the assay protocols as described in the manuscript. Following, analyze the oxygen consumption rate, measurement data, and the coupling efficiency data.

measurement-of-oxygen-consumption-rate-in-acute-mouse-brain-slices

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.1. Hydrate cartridge sensors (1 day before the assay)<ol><li>Open the extracellular flux assay kit (see&#160;Table of Materials) and remove both the sensor cartridge (green) and utility plate (clear;&#160;Figure 1A). Place the sensor cartridge aside (sensors up) and do not touch the sensors.</li><li>Add 600 &#181;L of calibrant solution (pH 7.4) to each well of the utility plate. Place the sensor cartridge on top of the utility plate and submerge the sensors in the calibrant solution. Make sure the triangular notch of the utility and sensor cartridge plate are correctly aligned (Figure 1B).</li><li>Seal the extracellular flux assay kit with sealing film to prevent evaporation of the calibrant solution, and then place it in a 37 &#176;C incubator not supplemented with CO2&#160;or oxygen overnight.</li></ol>2. Prepare the tissue plate (on the day of the assay)<ol><li>Open the islet capture microplate and take out the islet plate for tissue sitting.</li><li>Warm an appropriate volume (625 &#181;L per well) of pre-oxygenated modified artificial cerebrospinal fluid (ACSF, composition 140 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl2, 1.3 mM MgSO4, 0.3 mM KH2PO4, 25 mM glucose, 10 mM HEPES, pH 7.2) buffer to 37 &#176;C in a 50 mL tube. Then add BSA to a final concentration of 4 mg/mL to prepare the respiration buffer. In general, 50 mL is enough for one plate.</li><li>Add 625 &#181;L of respiration buffer (pre-oxygenated ACSF buffer with 25 mM glucose and 4 mg/mL BSA) to each well of the islet plate carefully and avoid shaking the plate. Ensure no residual drops are on top of the sensor cartridge and no air bubbles are present in the buffer of each well.</li></ol>3. Acute striatal slice preparation and excised slice placement<ol><li>Decapitate the mouse after cervical dislocation and immediately dissect the brain in 10 mL of ice-cold pre-oxygenated cutting solution (125 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 3.7 mM MgSO4, 0.3 mM KH2PO4, 10 mM glucose, pH 7.4).</li><li>Section coronal striatal slices with a vibratome following the manufacturer's instruction (see&#160;Table of Materials) at a thickness of 150 &#181;m in ice-cold, pre-oxygenated cutting solution.</li><li>Recover the slices in 50 mL of oxygenated ACSF (125 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 2.4 mM CaCl2, 1.3 mM MgSO4, 0.3 mM KH2PO4, 10 mM glucose, 2 mM HEPES, pH 7.4) and keep in solution for up to 30 min at room temperature (RT).</li><li>After recovery, transfer the slices to a 35 mm x 10 mm Petri dish with 5 mL of respiration buffer.</li><li>Use a stainless-steel biopsy punch (e.g., 1.5 mm diameter) to create a circular piece of tissue in the desired area of the sliced brain. Keep the slice in the buffer while gently pressing down with the punch. Make sure the punch is pushed down hard enough to cut the tissue. Remove the rest of the tissue, lift the punch away, and remove the circular piece of tissue into the buffer.</li><li>Cut the very end of a 1 mL pipette tip to make a hole with a 1.5-2.0 mm diameter and use it to hold and transfer the punched slice to the top of the capture screen. Suction one piece of punched brain tissue and carefully place the tissue onto the mesh side of the capture screen (Figure 2A). The capture screen will be a circular piece of plastic with the mesh attached to one side. NOTE: The capture screens are included in the microplate pack (see&#160;Table of Materials).</li><li>Gently use a paper tissue to dry the capture screen briefly. With the moisture removed, the tissue becomes sticky, which allows the slice to attach to the center of the mesh. Do not dry the slice too much, as it will be damaged otherwise.</li><li>Hold the capture screen slice side down with tweezers and place it into one of the wells of the incubating islet plate (Figure 2B). Be careful not to drop the slice; if so, take the screen out and place a new slice on it. NOTE: Do not place brain slices into the two background correction wells (A1 and D6) in the islet plate.</li><li>Incubate the islet plate at 37 &#176;C in an incubator for at least 30 min to allow temperature and pH equilibration before running the assay.</li></ol>4. Loading of cartridges with desired compounds<ol><li>Dilute the desired compounds in modified ACSF&#160;(37 &#176;C) to the final stock concentration of 10x, 11x, 12x, and 13x of the working concentration for ports A to D, respectively, and adjust to pH 7.4. The test will administer the compounds in order from port A to D. For this experiment, the following solutions were used, with their respective working concentration mentioned before them: 10 mM pyruvate (port A), 20 &#181;M oligomycin (port B), 10 &#181;M or other concentrations of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (port C), and 20 &#181;M antimycin A (port D).</li><li>Gently preload 75 &#181;L of the diluted compounds into the appropriate injection ports of the sensor cartridge. Place the tips halfway into the injection ports at a 45&#176; angle with the tip against the wall of the injection port. Tips should not be inserted completely to the bottom of the injection ports as this may cause compound leakage through the port.</li><li>Withdraw the tips from the ports carefully to avoid creating air bubbles. Do not tap any portion of the cartridge to avoid alleviating air bubbles.</li><li>Visually inspect the injection ports for even loading. Ensure all liquid is in the port and no residual drops are present on top of the cartridge.</li><li>Place the sensor cartridge onto the utility plate and put it into an incubator (non-CO2) for 30 min to allow it to heat up to 37 &#176;C again. Handle carefully by only holding onto the utility plate. Move as little as possible.</li></ol>5. Calibration and performing the assay<ol><li>Load the assay template in the software. Press the green&#160;START&#160;button.</li><li>Make sure to load the correct protocol. The protocol contains a combination of 3 min mix, 3 min wait, and 2 min measure sequences (these three steps form one measurement). Change the distance of the probe head from 26,600 to 27,800 in the hardware settings under instrument settings. There is no need to change the probe head for the XFe24&#160;analyzer. Press&#160;START.</li><li>Load the sensor cartridge on the utility plate into the instrument tray. The notch goes in the front left corner. Ensure that the plate sits correctly and is flat. Load the drug-filled sensor cartridge into the analyzer for calibration.</li><li>Follow the instructions on the screen to calibrate and equilibrate the sensors. This takes around 30 min.</li><li>Once the calibration step is done, remove the calibration plate and replace it with the islet plate (containing the mesh and tissue slices).</li><li>Measure the oxygen consumption in each well of the plate using the assay protocols. The protocol contains a combination of 3 min mix, 3 min wait, and 2 min measure sequences (these three steps form one measurement), at which point the oxygen consumption rate (OCR) is calculated.</li><li>For the entire experiment, include 4x OCR measurements to create a baseline, followed by the injection of port A (pyruvate) with 4x measurements, then the injection of port B (oligomycin) with 8x measurements, then the injection of port C (FCCP) with 5x measurements, and, finally, the injection of port D (antimycin A) with 6x measurements. NOTE: This number of measurements after each compound injection is determined depending on when the measurement reaches the plateau.</li><li>Analyze the OCR measurement data and the coupling efficiency data.</li></ol>

<figure class='table'><table class='ck-table-resized' style='border-collapse:collapse;font-family:Calibri;font-size:11pt;table-layout:fixed;' cellspacing='0' cellpadding='0' dir='ltr' border='1' data-sheets-root='1' data-sheets-baot='1'><colgroup><col style='width:25%;' width='459'><col style='width:25%;' width='258'><col style='width:25%;' width='182'><col style='width:25%;' width='395'></colgroup><tbody><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Accumet AB150 pH benchtop meter</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Thermo Fisher Scientific</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>13-636-AB150</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To measure pH</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Antimycin A from streptomyces sp.</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>A8674</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To inhibit complex III of the mitochondria</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Bovine Serum Albumin (BSA)</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>A6003</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To make modified artificial cerebrospinal fluid (BSA-ACSF)</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>C2920</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To uncouple mitochondrial respiration</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>D-Glucose</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>G8270</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To make artificial cerebrospinal fluid (ACSF)</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>DMSO</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>D8418</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To dissovle compounds</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>HEPES</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>H3375</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To make artificial cerebrospinal fluid (ACSF)</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Humidified non-carbon dioxide incubator</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Fisher Scientific</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>11-683-230D</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To hydrate plates at 37 &#176;C</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Oligomycin from Streptomyces diastatochromogenes</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>O4876</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To inhibit mitochondrial ATP synthase</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Parafilm</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA-ALDRICH</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>&#160;</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>sealing film</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse XF Calibrant Solution 500 mL</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse Bioscience</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>103681-100</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Solution for seahorse calibration</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse XF Extracellular Flux Analyzer</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse Bioscience</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>&#160;</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Equipment used to analyze oxygen consumption rate, old generation</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse XFe24 Extracellular Flux Analyzer</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse Bioscience</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>&#160;</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Equipment used to analyze oxygen consumption rate, new generation</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse XF24 FluxPaks</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Seahorse Bioscience</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>101174-100</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Package of flux analyzer sensor cartridges, tissue culture plates, capture screens,&#160; calibrant solution and calibration plates; assay kit.</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Sodium pyruvate</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>SIGMA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>P2256</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To prevent any substrate-limiting constraints of substrate supply</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Stainless steel biopsy punches</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Miltex</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>&#160;</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Device used to punch slices</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Sterile cell culture dish, 35 x 10 mm</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Eppendrof</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>30700102</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Used for slice punch</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Vibratome</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Leica</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>VT1200</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To slice brain tissue</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Water bath</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>Thermo Scientific Precision</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>282-115</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>To heat buffer and solutions</td></tr></tbody></table></figure>

<a href='https://app.jove.com/v/63379/measurement-oxygen-consumption-rate-acute-striatal-slices-from-adult'>Source: Zhi, L. et al., Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice. J. Vis. Exp. (2022).</a>This video demonstrates how to measure mitochondrial oxygen consumption rate (OCR) in mouse striatal brain slices. It details the steps for preparing the tissue plate and sensor cartridge, loading them into the flux analyzer, and explains the cellular processes underlying OCR patterns.

<figure class='image image-style-align-center image_resized' style='display:table;margin-left:auto;margin-right:auto;'><img style='aspect-ratio:2688/954;' src='https://cloudfront.jove.com/files/ftp_upload/23288/23288_Figure1_editor_23288.jpg' alt='23288_Figure1.jpg' /></figure>Figure 1: Images showing the extracellular flux analyzer hydrate cartridge sensors and plate.&#160;(A) The sensor cartridge sitting on top of a calibration plate (utility plate). (B) Side view of the utility plate and ...

Source: Zhi, L. et al., Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice. J. Vis. Exp. (2022).This video demonstrates how ...

Place brain slice punches from healthy and neurodegenerative mice on sample-holding screens.Flip the screens and insert them into a tissue plate.Take a pre-calibrated sensor cartridge with sensor probes. Load the compounds&#8212;pyruvate, oligomycin, FCCP, and antimycin A&#8212;into the injection ports.Place the cartridge on the tissue plate. Initiate the run in an extracellular flux analyzer.&#160;The probes measure the slice&#8217;s oxygen consumption rate or (OCR).With the first injection, pyruvate enters the mitochondria, generating electron carriers.These carriers donate electrons to Complexes I and II of the electron transport chain or (ETC), initiating an electron transfer reaction that increases oxygen consumption and OCR.Oligomycin inhibits Complex V, halting ATP synthesis and decreasing OCR.FCCP, a protonophore, disrupts the proton gradient, increasing oxygen consumption and OCR.Antimycin A binds to Complex III and stops the ETC, minimizing OCR.Compare the OCR profiles.A decreased OCR in the neurodegenerative slices indicates mitochondrial impairment.

7 ビュー. Measurement of Oxygen Consumption Rate in Acute Mouse Brain Slices

ビデオ: Measurement of Oxygen Consumption Rate in Acute Mouse Brain Slices - 実験

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

測定と酸素消費量全体の解釈飛ぶ頭セグメント

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

JoVE Journal

生化学

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

細胞外フラックス アナライザーを使用してプライマリの培養マウス新生児心筋酸素消費量の分析

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

医学

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

そのまま線虫の酸素消費量の測定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Measurement of Oxygen Consumption Rate in Acute Mouse Brain Slices

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Measurement of Oxygen Consumption Rate in Acute Mouse Brain Slices