Name: 视频: 使用细胞外通量分析仪实时监测人 NK 细胞中的能量代谢 - 概念
Uploaded: 2023-11-30T12:47:35.000+00:00
Duration: 1 min 35 s
Description: 118 次观看. 来源:Traba， J. et al.， 人类自然杀伤细胞代谢分析。J. Vis. Exp. （2020 年） 该视频演示了使用细胞外通量分析仪分析活化人 NK 细胞能量代谢的方法。电子传递链复合物的不同抑制剂会影响 ATP 合成，ATP 合成是通过耗氧率或 OCR 来衡量的，从而提供对细胞代谢和生物能量学的见解。

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			<td>2-Deoxy-D-glucose (2-DG)</td>
			<td>MilliporeSigma</td>
			<td>D8375-5G</td>
			<td>Glycolyisis stress test injector compound</td>
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			<td>2,4-Dinotrophenol (2,4-DNP)</td>
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			<td>Complex III inhibitor / glycolysis and mitochondrial stress test injector compound</td>
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			<td>BD FACSDIVA Software</td>
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			<td></td>
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			<td>BD Biosciences</td>
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			<td>EasySep Human CD3 Positive Selection Kit II</td>
			<td>Stemcell technologies</td>
			<td>17851</td>
			<td>NK cell isolation from PBMCs</td>
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		<tr>
			<td>EasySep Human NK cell Enrichment Kit</td>
			<td>Stemcell technologies</td>
			<td>19055</td>
			<td>NK cell isolation from PBMCs</td>
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			<td>EasySep Magnet</td>
			<td>Stemcell technologies</td>
			<td>18001</td>
			<td>NK cell isolation from PBMCs</td>
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			<td>EDTA 0.5 M, pH 8</td>
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			<td>Mouse anti-human CD56 PE</td>
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			<td>Mouse anti-human NKp46 PE</td>
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			<td>Oligomycin</td>
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			<td>Rotenone</td>
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			<td>Seahorse Wave Desktop Software</td>
			<td>Agilent</td>
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			<td>Sodium pyruvate (100 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11360-070</td>
			<td>Component of mitochondrial stress test medium</td>
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real-time monitoring of energy metabolism in human nk cells using an extracellular flux analyzer

Source: Traba, J. et al., <a href="https://app.jove.com/v/61466">Analysis of Human Natural Killer Cell Metabolism.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates the method to analyze energy metabolism in activated human NK cells using an extracellular flux analyzer. Different inhibitors of the electron transport chain complexes impact ATP synthesis, which is measured through the oxygen consumption rate or OCR, providing insights into cellular metabolism and bioenergetics.

Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Reagent preparation

	Reagents ...

Begin with an adhesive-coated assay plate containing a monolayer of activated natural killer cells.
Position a pre-calibrated sensor cartridge containing fluorophore-embedded sensor probes, surrounded by multiple injection ports.
Load various mitochondrial respiration inhibitory compounds into distinct ports of the cartridge.
In an extracellular flux analyzer, sensors measure the cells&#39; oxygen consumption rate, or OCR, with automatic compound injection.
In the first injection, oligomycin enters the cells&#39; mitochondria and interacts with complex V&#160; of the electron transport chain, or ETC, blocking ATP&#160; synthesis and decreasing OCR.
The second injection introduces 2,4-dinitrophenol &#8212; a protonophore that shuttles protons across the mitochondrial membranes, causing proton leakage. To restore the membrane potential, oxygen consumption increases, maximizing the OCR.
Finally, with the third injection, rotenone and antimycin A&#160; bind to ETC&#39;s Complex I and III, respectively, stopping ETC and decreasing OCR to its lowest level.
The real-time OCR profile with inhibitors reveals the energy metabolism of natural killer cells.

real-time-monitoring-energy-metabolism-human-nk-cells-using-an

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

118 次观看. 来源:Traba， J. et al.， 人类自然杀伤细胞代谢分析。J. Vis. Exp. （2020 年） 该视频演示了使用细胞外通量分析仪分析活化人 NK 细胞能量代谢的方法。电子传递链复合物的不同抑制剂会影响 ATP 合成，ATP 合成是通过耗氧率或 OCR 来衡量的，从而提供对细胞代谢和生物能量学的见解。 

视频: 使用细胞外通量分析仪实时监测人 NK 细胞中的能量代谢 - 概念

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

白念珠菌实时细胞外通量实时分析的生物能量学研究

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

科研

JoVE Journal

医学

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.

细胞外通量分析仪分析原代培养小鼠新生心肌细胞的耗氧率

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

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer

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Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer