这项工作部分是由国家科学基金会授予玛丽 e. Konkle 和迈克尔. Menze 的 CHE-160944 资助的。

1. 细胞培养
<ol><li>维持人肝癌 (HepG2) 细胞在 Dulbecco 的改良鹰的培养基 (DMEM) 含有10% 胎牛血清 (血清) 和附加基质 (10 毫米葡萄糖, 2 毫米谷氨酰胺, 1 毫米丙酮酸盐) 在37°c 在细胞培养孵化器 (5% CO2, 95% 空气, 100% 湿度)。 注: 在 respirometry 和量热仪的后续步骤中引用 DMEM 时, 请使用上述 DMEM 公式。</li>
	<li>将单元格在 1 x 106细胞每100毫米培养板上, 每7天或在到达90% 融合之前, 每 3-4 天更新培养基。</li>
	<li>在细胞 60-80% 汇合时进行实验。</li>
</ol>2. 呼吸和热量计的制备
<ol><li>吸管2.2 毫升的 DMEM 进入呼吸室, 充分插入塞子, 并调整与塞子间隔工具, 以使最佳的氧气扩散从空气到介质。</li>
	<li>连续搅拌37摄氏度, 直至氧浓度 (蓝迹) 和氧通量 (红迹) 稳定。确保呼吸的软件被编程来解释实验中所用介质的氧溶解度。 注: 不同介质有不同的氧溶解度系数 (例如, DMEM = 0.92)。</li>
	<li>在 DMEM 氧浓度稳定后, 用燃烧室打开, 选择氧浓度跟踪的稳定部分, 并校准100% 空气饱和度。</li>
	<li>校准0% 氧气选择稳定部分的氧气浓度的痕迹时, 没有氧气存在的房间。执行此步骤后, 20 µL 滴定230毫米硫酸钠钠或在所有氧气消耗的生物样品在实验结束。</li>
	<li>在100% 的空气校准呼吸, 关闭塞子, 并确保没有气泡出现在会议厅内。 注: 当极谱氧传感器 (POS) 消耗氧气以测量氧分压时, 密闭腔内的氧通量会轻微增加。</li>
	<li>在10分钟的塞子被关闭, 确认呼吸是准备 HepG2 细胞通过观察稳定的氧气流量。 注: 这里使用的热量计采用不锈钢安瓿, 需要一个安瓿作为参考。</li>
	<li>将2.5 毫升的 H2o (dH2o) [相等体积作为样本 (步骤 4)] 添加到热量计的参考安瓿并拧紧瓶盖, 如有必要, 使用钳子。清洁密封安瓿与气流, 并慢慢降低安瓿到位置1在参考通道的热量计。保持在位置1直到生物样品准备好。</li>
</ol>3. Respirometry 和量热法制备 HepG2 细胞
<ol><li>确认呼吸和热量计的制备和校准。温暖的 DMEM 和胰蛋白酶到37°c 在水浴。</li>
	<li>添加 DMEM 到一个新鲜的100毫米板和放置在孵化器, 以允许平衡的媒介为 CO2和温度。 注: 此介质将用于量热实验, 因此适当的体积取决于将使用的安瓿的数量。</li>
	<li>用倒置光显微镜确认细胞在 60-80% 汇合处, 然后用10毫升室温磷酸盐缓冲盐水 (PBS) 洗涤100毫米板2x。</li>
	<li>添加3毫升的37°c 胰蛋白酶到100毫米细胞和孵化他们为 7-10 分钟在37°c 在细胞培养孵化器。将胰蛋白酶与3毫升的37°c DMEM 中和, 通过轻轻吹打20倍于5毫升吸管。</li>
	<li>将 DMEM 和胰蛋白酶中的6毫升悬浮细胞转移到无菌的15毫升圆锥管中, 在 175 x g 处将其离心为5分钟. 取出液体而不干扰细胞颗粒。</li>
	<li>并用重悬细胞颗粒在 ~ 5 毫升的 DMEM 和吸管它彻底地确保细胞彼此充分分离。</li>
	<li>删除100µL 的混合样本, 并使用 hemocytometer 上的所有8个字段进行彻底的单元格计数, 以确定单元格的总数。然后计算单元格泥沙的音量, 以便每50µL 生成 ~ 2 x 106单元格。 注意: 这将确保将 2 x 106单元格添加到呼吸的每个分室中, 且介质位移最小。</li>
	<li>将细胞离心5分钟, 在 175 x g 并用重悬从步骤3.7 中计算出的 DMEM 量中的颗粒。</li>
	<li>执行单元格计数以确定在呼吸中每室注入 2 x 106单元格所需的卷 (这应该是 ~ 50 µL)。储存在冰上的 respirometric 测量的细胞, 直到准备就绪。</li>
	<li>当浓缩的细胞样品在冰上时, 确定稀释量为 1 x 105细胞/毫升的热量。</li>
	<li>将样本进行混合, 并在每个安瓿的 DMEM 中为 1 x 105细胞/毫升准备3毫升的细胞。 注: 样品稀释用的 DMEM 应为步骤3.3 中制备的平衡培养基。</li>
</ol>4. 量热法
<ol><li>确保热量计与计算机连接, µW 中的热信号是可观测和稳定的。</li>
	<li>将2.5 毫升的细胞在 1 x 105细胞/mL (~ 2.5 x 105细胞) 中添加到每个安瓿, 确保足够的空气在安瓿和介质的盖子之间, 以允许气体扩散。</li>
	<li>立即拧紧瓶盖, 必要时使用钳子。清洁密封安瓿, 使用气流去除任何外部碎片, 并慢慢降低安瓿的位置1的热量计。记录安瓿降低到位置1的时间。</li>
	<li>允许安瓿在位置1处坐15分钟。 注意: 在这15分钟期间, 细胞将被注射入呼吸 (步骤 5)。这确保在确定 CR 比时, 同样的时间间隔用于热量生产和氧气消耗。</li>
	<li>15分钟在位置 1, 慢慢地降低参考和样品安瓿到位置2。记录安瓿降低的时间, 并测量至少30分钟。</li>
</ol>5. Respirometry
<ol><li>在15分钟间隔内, 安瓿在热量计中的位置 1, 开始 respirometric 研究。</li>
	<li>通过吹打彻底混合细胞样本, 以确保一个均匀的溶液, 并注入 < 50 µL 细胞样本到呼吸的每个腔内, 最终浓度为 ~ 2 x 106细胞/室。记录单元格注入呼吸的时间。</li>
	<li>注射后, HepG2 细胞连续搅拌37摄氏度。至少等待 20-30 分钟的氧气流量稳定和氧浓度的稳定下降。</li>
	<li>选中屏幕右侧的O2每 V 的通量选项卡, 并突出显示氧气流量的稳定部分。选择标记, 然后统计和记录 O2通量 (每 v) 的数据。此记录将用于计算 CR 比率。 注意: 步骤5.5 和5.6 是可选的。寡霉素和 FCCP 的滴定揭示了漏呼吸和最大不耦合呼吸的情况。</li>
	<li>注入1µL 4 毫克/毫升寡霉素到每个房间, 并允许〜10分钟的氧气流量稳定。通过以下步骤记录稳定的漏呼吸率5.4 通过突出一个稳定部分的氧气流量的痕迹后寡霉素添加。</li>
	<li>滴定 2-µL 注射0.2 毫米 FCCP 进入每个室, 允许氧通量稳定后每次注射。继续滴定直到达到最大通量, 如随后的 FCCP 滴定略有下降所示。在最大通量期间记录稳定的呼吸速率。</li>
</ol>6. Calorespirometric 比的计算
<ol><li>对使用 hemocytometer 的所有8个字段的 1 x 105单元格/mL 中准备的样本进行最终单元格计数, 以确定添加到安瓿 (2.5 mL) 中的单元格总数。</li>
	<li>确定一个时间来记录测量, 从热量计和呼吸, 稳定的信号在同一时间存在。参考当安瓿被降低到位置2和注入细胞进入呼吸时记录的时间。</li>
	<li>通过将光标放在显示热量输出的迹线上, 在步骤确定的时间和表达为µW/百万细胞时, 记录热生产。收集氧气消耗数据, 并确保它被表示为 pmol O2 x s-1 x 万元单元格。</li>
	<li>要计算 CR 比率, 首先将µW 转换为焦/秒, 然后除以 O2/秒的摩尔上的焦/秒, 以获取表示为焦/摩尔 O2的 CR 比率。注意: CR 比率在焦/摩尔 O2中显示。 (请参见<a href="/files/ftp_upload/57724/JoVE_Supplemental_File.docx" target="_blank">辅助文件 1</a>)</li>
</ol>

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作者没有什么可透露的。

calorespirometry 的目的是定量评价有氧和厌氧途径对代谢活动的贡献, 并获得细胞能量通量的综合观点。这是通过同时测量散热和氧通量, 然后用计算的 CR 比与理论 oxycaloric 当量的比较来完成的。对于可重现和可靠的数据, 必须考虑几个关键步骤。维持一种无菌、健康的细胞培养是至关重要的。细菌或真菌污染可能导致被挪用的热量生产或氧气消耗量, 使 CR 比率没有意义。此外, 细胞计数不当, 样本混...

在生物系统中, 新陈代谢过程中的热释放或焓变化通常直接通过 O 2消耗量和/或 CO2 (通过 respirometry) 来监测。不幸的是, 当这些技术被隔离使用时, 关键信息就会丢失, 例如厌氧途径对细胞代谢的贡献。Calorespirometry 是一种强大的技术, 依赖于同时测量散热和呼吸。开创性的 calorespirometric 工作研究了完全氧合哺乳动物细胞的厌氧代谢, 并证明了有氧和厌氧途径同时贡献能量动态平衡, 尽管转化的细胞在完全氧合环境1。Calorespirometry 已经被应用到各种各样的生物学问题上。一些例子包括研究低氧水平的动物热力学, 除草剂和雌激素对双壳贝类鳃的影响, 陆地有机体的新陈代谢, 以及有机土壤物质的微生物分解2,3,4,5,6. 此外, calorespirometry 还揭示了冷冻前的代谢预处理改善了哺乳动物细胞的冷冻保存7。每种方法, 无论是量热和 respirometry, 都独立增加了我们对细胞和生物体生物能学的认识。然而, 可以通过使用 calorespirometry 来回答的基本生物学问题仍然是相对未探索的。
赫斯定律指出, 反应的总焓变化与初始和最终状态之间的通路无关。例如, 生化通路的总焓变化是通路内所有反应焓的变化总和。量热法提供了一种测量细胞热量的实时方法, 它不加区别地检测有氧和厌氧通路。这是基于没有在系统中交换能量的基础, 除非通过实验安瓿8的墙壁。散热的变化等于安瓿中所有代谢反应释放出的焓的变化。因此, 负焓与系统的热量损失相关。在过去四年里, 详尽的研究描绘了分解代谢和代谢的热力学景观。这是由美国国立医学图书馆 (NLM) 在国家卫生研究院 (PubMed) 索引的 "生物" 和 "量热" 搜索词下的研究文章的稳步增加所代表的。搜索结果显示, 在1970之前, 共有27份出版物参考生物量热仪;与此同时, 仅在 2016年, 就有546份出版物利用了这项技术。
热方法的建立, 以确定热生产。但是, 对于解决 respirometric 值, 还是给予了更大的灵活性。respirometric 度量可以由 o2、CO2或 o2和 co2组成。此外, O2或 CO2的测量可以通过各种技术完成, 其中包括 optrodes、克拉克型电极和可调谐二极管激光吸收光谱7、9、10 ,11。虽然 CO2生产在许多 respirometric 研究中是一个有价值的指标, 但培养细胞的培养基通常利用 bicarbonic 缓冲系统来控制 pH 值1213。为避免在碳酸氢钠系统中 CO2测量的并发症, 以下用于区域性单元格 calorespirometry 的协议利用 O2作为唯一 respirometric 参数。
在测量氧通量的同时, 某些 respirometers (参见材料表) 是为了详细评估线粒体功能而设计的。衬底-uncoupler 抑制剂-滴定 (套装) 协议是很好的建立和兼容的实验设计, 以测量膜电位或活性氧 (ROS) 形成14。所提出的完整细胞 calorespirometry 协议与化学 uncouplers 的引入相兼容, 如羰基氰化物-trifluoromethoxyphenylhydrazone (FCCP) 和 f0f1-ATP 合成酶抑制剂寡霉素。通过添加 FCCP, 氧消耗可以从 ATP 生产中拆解, 这有助于评估潜在疗法对线粒体绩效15的影响。此外, 寡霉素的添加也照亮了泄漏呼吸的程度。因此, 在 calorespirometry 期间进行的 respirometric 测量与广泛的协议兼容, 旨在进一步阐明线粒体生理学。
同时测量散热和氧通量可以计算 calorespirometric (CR) 比。这个比率然后被比较对桑顿的恒定或理论 oxycaloric 等值, 范围在-430 到-480 焦摩尔-1之间根据细胞线或组织利益和被补充的碳基体1, 16. 因此, 一个更负的 CR 比率揭示了从厌氧途径到整体新陈代谢活动的贡献增加。例如, 常规肌肉组织呼吸的 CR 比率没有工作的活动性能范围从-448 到-468 焦 mol-1, 这是在理论 oxycaloric 等效17,18的范围内。同时, 在高葡萄糖的培养基中培养的哺乳动物癌细胞, 在糖酵解和相对低线粒体接触19的情况下增强了乳酸发酵。这种表型结果在-490 至-800 焦摩尔-1范围内的 CR 比值, 显示了更多的负 CR 比值1,7, 在细胞代谢中的厌氧通路的增加参与, 16,20。
商业和非营利细胞和组织分销商目前提供150多个物种的细胞线, 近4000个细胞系来源于人类。永生化细胞系是快速评估潜在疗法毒性的方便工具, 其中许多可能直接或间接干扰线粒体功能。在药物筛选过程中使用转换后的细胞可能具有有限的预测价值, 部分原因是因为华宝效应是许多癌症的标志。通常, 癌症产生 ATP 从基质层磷酸化和保持氧化还原平衡, 通过生产乳酸, 而不充分参与线粒体下有氧条件下19。制药业的发展是众所周知的昂贵和低效的, 约8的9化合物测试在人体临床试验未能达到市场批准21。虽然潜在的治疗可能通过初步筛选由于细胞株的细胞毒性低, 有可能这些化合物中的一些 mitotoxic。如果没有一个合适的方法来检测这些毒素如何损害不显示华宝效应的初级细胞的能量平衡, 关键信息往往是过度观察, 瓶颈治疗发展的早期阶段。
Calorespirometry 是一种实用的、无创的方法来分析各种生物样品中的代谢活动, 包括细胞和组织。所提出的协议的核心与一系列应用程序兼容。然而, 已经发现了一个并发症。永生化细胞通常培养在无葡萄糖培养基中补充半乳糖, 以增加氧化磷酸 (OXPHOS) 对能源生产的贡献, 以便使细胞敏感到潜在的 mitotoxins22, 23。当样品放在热量计15使用的不锈钢安瓿中时, 这种代谢重新编程似乎不清楚分析。在葡萄糖培养基中培养的细胞继续从事高代谢活动几个小时。同时, 在半乳糖培养基中培养的细胞在安瓿的30分钟内减少了热量的产生, 使测量仅限于早期实验时间点。不幸的是, 这种行为阻碍了评估细胞增殖的机会。尽管有这一特定限制, 大多数应用都与 calorespirometric 分析兼容, 并且可以通过这种方法获得详细的代谢信息。

<table><tbody><tr><td>HepG2 Cells</td><td>American Type Culture Collection</td><td>HB-8065</td><td>Cells used for calorespirometry</td></tr><tr><td>O2k-Respirometer</td><td>Oroboros Instruments</td><td>10022-02</td><td>Respirometer</td></tr><tr><td>LKB 2277 thermal activity monitor (TAM)</td><td>Thermometric AB</td><td></td><td>Thermometric was purchased by TA Instruments</td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Thermofisher Scientific</td><td>11360070</td><td>100x solution added to DMEM medium</td></tr><tr><td>Fetal Bovine Serum - Premiuim Select</td><td>Atlanta Biologicals</td><td>S11550</td><td>Added to 10% in DMEM medium</td></tr><tr><td>Trypsn-EDTA (0.25%)</td><td>Thermofisher Scientific</td><td>25200072</td><td>Cell dissociation reagent</td></tr><tr><td>Oligomycin from &lt;em&gt;Streptomyces diastatochromogenes&lt;/em&gt;</td><td>Sigma Aldrich&amp;nbsp;</td><td>O4876</td><td>Mitochondrial Inhibitor</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td><td>Sigma Aldrich</td><td>C2920</td><td>Mitochondrial Uncoupler</td></tr><tr><td>Corning 100 mm TC-Treated Culture Dish</td><td>Corning Corporation</td><td>430167</td><td>Tissue culture dish</td></tr><tr><td>Glucose, powder</td><td>Thermofisher Scientific</td><td>15023021</td><td>Glucose for DMEM medium</td></tr><tr><td>Galactose, powder</td><td>Fischer Scientific</td><td>BP656500</td><td>Galactose for DMEM medium</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Thermofisher Scientific</td><td>25030081</td><td>Glutamine for DMEM medium</td></tr><tr><td>DMEM, no glucose</td><td>Thermofisher Scientific</td><td>11966025</td><td>Cell culture medium</td></tr></tbody></table>

calorespirometry: a powerful, noninvasive approach to investigate cellular energy metabolism

在基本生物和生物医学研究中使用的许多细胞系通过有氧和厌氧呼吸的结合来维持能量平衡。然而, 这两种途径在多大程度上促进了细胞能源生产的前景一直被忽视。在饱和葡萄糖中培养的转化细胞往往会减少对 ATP 生产的氧化磷酸化的依赖性, 这是由于基质层磷酸化的增加而得到补偿的。这种新陈代谢平衡的转变允许细胞增殖, 尽管存在线粒体毒素。在忽视转化细胞的新陈代谢平衡时, 药物筛选的结果可能会被误解, 因为在高葡萄糖存在下培养的模型细胞株可能无法检测到潜在的 mitotoxic 效应。浓度。该协议描述了两种强大的技术, respirometry 和量热, 这使得对细胞 ATP 生产的有氧和厌氧贡献的定量和无创评估。有氧和厌氧呼吸产生热量, 可以监测通过量热。同时, 测定氧耗率可以评估有氧呼吸的程度。当同时测量散热和耗氧量时, 可以确定 calorespirometric 比。实验得到的值可以与理论 oxycaloric 等效, 可判断厌氧呼吸的程度。因此, calorespirometry 提供了一种独特的方法来分析广泛的生物学问题, 包括药物的发展, 微生物的生长, 和基本生物能学在常氧和缺氧条件下。

calorespirometric 测量的重现性取决于适当和一致的样品准备。如果板块生长过度, 细胞培养所制备的样品不应使用, 因为细胞计数可能由于结块而变得不准确。此外, 由于在丛生细胞中的基底扩散有限, 可能会发生热量流动减少。因此, 在使用粘附细胞时, 选择一个融合在 60-80% 之间的板并在实验之前改变中等 24 h 是至关重要的。
适当的仪器校准和维护对于信息和可重现的 calorespiro...

Watch this Scientific Journal Video about Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism at JoVE.com

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

该协议描述 calorespirometry, 直接和同时测量散热和呼吸, 这提供了一种无创的方法来评估能量代谢。该技术用于通过监测总的细胞能量流来评估有氧和厌氧途径对能源利用的贡献。

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

calorespirometry-powerful-noninvasive-approach-to-investigate

Research

JoVE Journal

Bioengineering

7.5K 次观看.  University of Louisville. 该协议描述 calorespirometry, 直接和同时测量散热和呼吸, 这提供了一种无创的方法来评估能量代谢。该技术用于通过监测总的细胞能量流来评估有氧和厌氧途径对能源利用的贡献。

视频: Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

使用C2C12成肌细胞的生物能简介实验

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

生物学

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

实时测量中性粒细胞的线粒体生物能量特征

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

免疫与感染

<meta charset="utf8"></head><body>乙醇通过转向谷氨酰胺依赖性呼吸来损害 AM 生物能量学

Assessment of Glutamine as a Fuel Source for Alveolar Macrophages Exposed to Chronic Ethanol Using an Extracellular Flux Bioanalyzer

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use impairs the ability of AMs to phagocytize and clear pathogens from the alveolar space, in part through dysregulated fuel metabolism and bioenergetics. Our prior work has shown that chronic ethanol (EtOH) consumption impairs mitochondrial bioenergetics and increases lactate levels in AMs. Further, we recently demonstrated that EtOH increases glutamine dependency and glutamine-dependent maximal respiration while decreasing flexibility, shifting away from pyruvate-dependent respiration and towards glutamine-dependent respiration. Glutaminolysis is an important compensatory pathway for mitochondrial respiration when pyruvate is used for lactic acid production or when other fuel sources are insufficient. Using a mouse AM cell line, MH-S cells, exposed to either no EtOH or EtOH (0.08%) for 72 h, we determined the dependency of mitochondrial respiration and bioenergetics on glutamine as a fuel source using an extracellular flux bioanalyzer. Real-time measures were done in response to bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), an inhibitor of glutaminase 1, which prevents the enzymatic conversion of glutamine to glutamate, in media vehicle or in response to vehicle alone, followed by testing mitochondrial stress. The step-by-step protocol provided herein describes our methods and calculations for analyzing average levels of glutamine-dependent basal mitochondrial respiration, mitochondrial ATP-linked respiration, maximal mitochondrial respiration, and mitochondrial spare respiratory capacity across multiple biological and experimental replicates.

<meta charset="utf8"></head><body>使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

Alcohol misuse impairs alveolar macrophage (AM) immunity due to suppressed mitochondrial respiration and bioenergetics. We recently demonstrated that ethanol (EtOH) exposure increases glutamine dependency for mitochondrial respiration in AMs. Herein, methods are provided to determine the usage of glutamine for mitochondrial respiration in EtOH-treated AMs using an extracellular flux bioanalyzer.

使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

高分辨率呼吸测定法，使用基于腔室和板式呼吸计评估细胞和组织中的生物能量学

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

使用高分辨率呼吸测定法实时分析原代人视网膜色素上皮细胞中的生物能量学

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

摘要

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使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

使用C2C12成肌细胞的生物能简介实验

实时测量中性粒细胞的线粒体生物能量特征

使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

高分辨率呼吸测定法，使用基于腔室和板式呼吸计评估细胞和组织中的生物能量学

使用高分辨率呼吸测定法实时分析原代人视网膜色素上皮细胞中的生物能量学

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

摘要

探索更多视频

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

使用C2C12成肌细胞的生物能简介实验

实时测量中性粒细胞的线粒体生物能量特征

使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

使用细胞外通量生物分析仪评估谷氨酰胺作为暴露于慢性乙醇的肺泡巨噬细胞的燃料来源

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

高分辨率呼吸测定法，使用基于腔室和板式呼吸计评估细胞和组织中的生物能量学

使用高分辨率呼吸测定法实时分析原代人视网膜色素上皮细胞中的生物能量学

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞