Name: measurement and analysis of extracellular acid production to determine glycolytic rate
Uploaded: 2015-12-12T15:00:00.000+00:00
Duration: 6 min 47 s
Description: Watch this Scientific Journal Video about Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate at JoVE.com

We thank David A. Ferrick and David G. Nicholls for contributing to project conception and presentation, Renata L.S. Goncalves and Akos A. Gerencser for data not shown here and for helpful discussions, Barbara Liepe for XF24 consumables, and Andy Neilson for input in developing Eq. (5).

1.测量缓冲输出功率是在细胞外流量仪表:两个变化注:计算与这里描述的方法是使用一个外磁分析器开发的。对于其他仪器，测量体积一定会适当缩放。 <ol><li>准备0.1M的盐酸标准使用盐酸浓缩水（见材料及设备），根据制造商的说明。 注意:一个例子计算了制备盐酸注射在所有4个注射口使用示于表1: </li></ol><img alt="表格1" src="/files/ftp_upload/53464/53464table1.jpg" /> 表1.盐酸连续注射到细胞外流量分析孔。 <ol start="2"><li>制备HCI标准的稀释液中的介质将在表1中的技术复制是卡尔数测定作为灭蝇灯在一个盘子，还有一个让移液错误;例如，为港口的四个技术重复A注射，准备（1.1微升×5），0.1 M盐酸股票在（48.9微升×5）分析培养基。 </li><li>分发50微升到测量探头盒的每个端口一个。重复此过程，其余端口B，C和D. </li><li>运行的一个细胞外磁通测定10具有标准校准周期，接着[2分钟的混合，1分钟的等待，和5分钟测量]每个四端口相加的两个周期（ 参见图2）。 <ol><li>根据软件指令编程实验以上在仪器的软件。装入准备好的墨盒插入机器，并按照软件的指示进行校准。 </li><li>当程序提示，取出含校准板，插入包含在每孔放入仪器检测培养基中板;继续运行程序。 </li></ol></li><li> ü<html>从之前的唱在稳态获得8-10个数据点（通常是最后8-10分）的平均值和每个端口加入后，计算在pH值（ΔpH）所引起的各喷射标准酸的（累积）差。 </li><li>绘制ΔpH对纳摩尔H +包含在7微升量被困测量探头。直线的斜率是英里/皮摩尔H +缓冲电源（BP）。 </li><li>另外的步骤1.2-1.3，开展ΔpH测量以下其中端口A，B和C被用于进行实验的分析，然后是盐酸注射端口D. 如表2，四大技术复制是用于产生在细胞外磁通测定板的20实验孔（不包括四背景温度校正孔）的每个点5点标准曲线。 </li></ol>53464table2.jpg"/&gt; 表2.单盐酸注入到细胞外流量分析孔。 2.测量使用外部pH计缓冲电源注意:为了测量的介质的使用外部pH探头缓冲功率，校准探头，在37℃，在试验过程中保持该温度下的所有试剂。 <ol><li>根据制造商的说明书使用盐酸精矿准备0.1M的标准盐酸水。 </li><li>暖pH探头，pH值的标准，其缓冲功率是要测量的分析培养基，和0.1M HCl中和至37℃的水浴中。 </li><li>根据制造商的说明书进行校准pH探头在37℃。通过使用热板或水浴保持整个测定所有试剂在37℃。 </li><li>等分试样将10ml测定培养基的入一个小烧杯或锥形管。监测pH值连续使用浸入pH探头。 </li><li>加入0.1 M盐酸到作为说媒体在10-20微升等份。 <ol><li>确保用搅拌棒或通过手动旋转的容器各酸加入后搅拌。 </li><li>允许几秒钟的pH测量来稳定，然后记录每次加入后的pH。 </li><li>这表现在表3中 ，使添加有足够数量，以保证准确的斜率计算，并覆盖在实验过程中预期的pH值范围。 </li></ol></li></ol><img alt="表3" src="/files/ftp_upload/53464/53464table3highres.jpg" width="650" /> 表3.使用pH计测量缓冲力。数据代表一个典型的实验中具有0.1 M盐酸6 20微升添加。 <ol start="6"><li>情节ΔpH针对纳摩尔H +的每7微升加入，赋予表示缓冲功率（图1）的线性斜率。 </li></ol> 图1.确定缓冲能力。 HCI标准曲线测定，如表1，表2或（这里） 如表3中。线性曲线拟合的斜率给出了缓冲功率（pH值/纳摩尔H +在7微升）。每个点代表意思的n±SEM = 9技术复制。 <ol start="7"><li>一旦缓冲功率通过方法1或2以上已知的，通过将ECAR通过缓冲功率转换ECAR信号（MPH /分钟）到PPR（皮摩尔H + /分钟/微克蛋白）（BP）（MPH /皮摩尔H +）和缩放到的每个孔中的蛋白质含量: PPR TOT（皮摩尔H + /分钟/微克蛋白）= ECAR（MPH /分钟）/ BP（MPH /皮摩尔H + 7微升），每孔（微克）/蛋白方程6 </li><li>另外，使用方法相同的实验第1条或2来计算仪器所使用的软件，数据采集过程中，自动计算PPR的缓冲能力（BC）值。 注:本仪器用户手册12（第107页）提供了有关计算和使用的缓冲能力，其中BC被描述为详细信息 BC（摩尔/升）=摩尔的 H + /（ΔpHX缓存容积（L））等式7 注:在等式7所述的缓冲能力作为可以计算在上述的仪器或外部pH探头测定。缓冲能力和缓冲能力之间的转换是很容易做到（见附件表格）: BC = 1×10 -9 / BP（（MPH /皮摩尔H + 7微升）/ 7微升） 方程8 注意:如果事先在执行测定已知，缓冲能力，可以直接输入到实验设置在仪器的软件。 </li><li>应用此程序及以上用于最传统的缓冲系统的计算，如前公布6描述。 注: 表4列出了缓冲力和几个常规介质的缓冲能力。 </li></ol><img alt="表4" src="/files/ftp_upload/53464/53464table4.jpg" /> 表4.缓冲能力和所选媒体的缓冲能力。 3.执行外通量测定使用C2C12成肌细胞注:在步骤3.4.3，有在CO 2没有观察到的差异衍生酸的生产依赖于碳酸酐中的C2C12培养的存在下，这表明不要求其存在的CO 2完全转化为HCO 3 - + H +。但是，实证检验这在不同的实验系统之前OM建议itting碳酸酐酶。 <ol><li>培养小鼠C2C12在Dulbecco改良的Eagle与11.1毫米的葡萄糖，2mM谷氨酰胺，10％体积/体积的胎牛血清（FBS），100U培养基（DMEM）成肌细胞13，在37℃下95％空气/ 5％CO 2 / ml青霉素和100μg/ ml链霉素。 </li><li> 24小时在测定前，板/种子细胞于100μl相同培养基中以20,000细胞/孔在24孔聚苯乙烯外通量测定板（参见材料和方法），没有附加的涂层。 </li><li>稀寡，FCCP，和鱼藤酮加myxothiazol和HCl（可选），以10倍终浓度克雷布斯林格氏磷酸盐HEPES（KRPH）分析培养基（2毫米的HEPES，136毫米氯化钠，2.5毫米的NaH 2 PO 4，3.7毫米氯化钾，1毫氯化镁 ，1.5mM的氯化钙 ，0.1％w / v的脂肪酸的牛血清白蛋白，pH 7.4中在37℃下）。 </li><li>细胞制备<ol><li>在测定前30分钟，通过吸向戈洗涤贴壁细胞三次ntly并去除培养基，然后慢慢加入500微升KRPH。 </li><li>在37℃下的第三次洗涤步骤在空气中温育细胞（未在5％ 的 CO 2，这将改变这个碳酸氢盐的培养基的pH）。 </li><li>在试验开始时，在孔用500μl含有500U / ml的碳酸酐酶和任葡萄糖（10毫米）或仅培养基新鲜KRPH替换KRPH，没有额外的基底。 </li></ol></li><li>加载传感器盒<ol><li>端口A:2微克/毫升寡，端口B:0.5μMFCCP，端口C ++编写的步骤3.3成外流量传感器盒如下（最终浓度测定孔中给出）的盒式磁带端口每10倍的化合物吸取50微升等份:1μM的鱼藤酮，1μM的myxothiazol，端口D:盐酸（如果上文和表2中所描述的执行中，测定酸校准）。 注:为完整的呼吸链的抑制目的这里描述为1μm我xothiazol可以互换使用以1μM的抗霉素A. </li></ol></li><li>外流量分析: <ol><li>确定呼吸控制在10描述执行标准外通量检测。 </li></ol></li></ol>注:对于实验的每一段，确定组合，等待，测量时间需要，每段周期以及数量。 注意: 在表5中的数据 ，收集了2分钟的混合，1分钟的等待，和5分钟测量每个区段的两个测定周期，与发生的端口D添加不同量的HCl（的用于缓冲的校准后3测定周期功率如表2中）。 <img alt="表5" src="/files/ftp_upload/53464/53464table5.jpg" /> 表5.外流量分析配置。 4. MeasuriNG终点乳酸浓度注意:要通过测量还原的初始速度（超过2分钟）验证在细胞外通量实验可以直接在常规的96孔板测定结束了描述在一些不同的系统，终点乳酸浓度的间接测定法NAD +→NADH催化乳酸脱氢酶，详细在我们先前的出版物6所述。在代表结果呈现的数据，在含有葡萄糖的测定孔的终点乳酸浓度为〜40微米。 <ol><li>制备2×肼介质:的1M Tris，20毫摩尔EDTA，400毫肼，pH值9.8在22℃）。立即测定开始前，加入NAD +至4毫和乳酸脱氢酶（LDH），以40单位/毫升。最终测定介质组合物（1×）:500毫摩尔Tris，10mM的EDTA，200mM的肼，2mM的NAD +，20U / ml的LDH。 </li><li>紧随外流量分析，去掉100微升从细胞外通量测定板，并转移到一个不透明（黑）的阱的每个孔的测定培养基的96孔板的。 </li><li>每个样品均匀，加入100微升2倍肼网上平台。 </li><li>立即加载板成酶标仪，并开始监测NADH的荧光在340nm激发/ 460nm发射。 </li><li>记录初始速度为约2分钟。 </li><li>运行一个类似的实验中通过标绘对乳酸浓度初始速度为加入乳酸浓度为0至50μM的构建标准曲线。 </li><li>计算乳酸浓度在每个实验使用标准曲线井。 </li></ol> 5.测量蛋白质含量<ol><li>从测定板的每个孔中除去剩余的测定培养基。 </li><li>洗孔三次用250微升BSA无KRPH，小心翼翼地尽量减少细胞从井底面松脱。 </li><li>加入25微升RIPA裂解介质（150毫摩尔NaCl，50毫摩尔Tris，1mM的EGTA，1mM的EDTA，1％体积/体积的Triton X-100，0.5％w / v的脱氧胆酸钠，0.1％体积/体积的SDS，pH 7.4中在22℃C）的到测定板的各孔中。 </li><li>孵育板置于冰上30分钟。 </li><li>搅动板在板振荡器上在1,200rpm下5分钟。 </li><li>测量蛋白质浓度通过标准方法，例如，通过BCA测定，确保裂解缓冲液组合物是与测量方法相兼容。在实验中图 2中的蛋白质含量为〜4微克/孔。 </li></ol>

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绍纳Mookerjee博士宣称，她已经没有竞争的财务权益。马丁品牌博士曾征询海马生物科学，其产生本条仪器和试剂。由海马Biosciences公司支付的对这篇文章开放存取费用。

细胞外酸化是细胞代谢率的容易衡量指标。为了正确地确定细胞糖酵解的速率（由乳酸生成定义）关键是要知道的测定培养基的缓冲能力，并可将氧消耗和酸化的胞外通量测量到质子生产率。通过进行该计算，从CO 2释放在柠檬酸循环造成的酸化可被减去，留下所导致乳酸产生的酸化。 这里给出以测量缓冲功率为这种校正的多种不同的方式携带不同的优点和缺点。使用pH探头外部测量是非常准确和可重复的，但可能不反映在由包含在测定板中，测定过程中加入的化合物，或叔的存在内的荧光团引入pH值检测小的差异他细胞本身。内的板的pH测量解决这些问题，还介绍了不同程度的实验噪声。 CO 2的校正ECAR允许首次糖酵解速率的明确的和定量的计算，和一个实验的过程中，揭示了变化，以总ECAR呼吸道和糖酵解的贡献。使用公式5和OCR，ECAR的测量，和缓冲力，糖酵解速率可以使用所提供的简单的电子表格（表6）来计算。此速率可以通过事后乳酸盐测量如果需要6进行验证。在细胞中，其中所述戊糖磷酸途径是高活性的，使用途径抑制剂如6- aminonicotinamide的可能是有用的隔离糖酵解速率。测定和乳酸衍生的 H +从总细胞外酸化速率和氧Consumpt -计算两者的 CO 2的贡献离子率是一个非常重要的工具使用外流量数据来做出关于代谢活性强大的和定量的语句。 使用此处描述的方法，其中包括用于测定缓冲功率，优化外通量实验下调查和所需的数据的单元的各种修改，糖酵解在完整细胞中的速率可以在大范围的实验条件下进行定量。这种方法仅限于细胞，可以在贴壁培养上生长（或细胞或细胞器可以粘附到）的聚苯乙烯表面。当培养的细胞是同质的并汇合它是最可靠的，尽管有用的数据仍然可以在一定范围的这些条件来获得。计算所需要的细胞的代谢的一些知识，作为最大的 H + / O 2的范围是从0.65到1.0的不同基板多为部分氧化6的充分氧化，但是，如果细胞k个nown氧化葡萄糖，可以假定的1.0的值。 虽然有关的所有代谢的表征，此方法可以在系统中使用时是特别有用的，其中呼吸道和糖酵解代谢之间的位移，以保持细胞ATP供给是一个​​关键的表型，包括干细胞和肿瘤衍生癌细胞的表征。理解这些和其他情况下的代谢控制的改变将允许在实验设计这些细胞类型的分析和更大程度的复杂性和准确性。

该方法的总的目标是精确地测量用外通量分析细胞的糖酵解速率。使用外酸化糖酵解率的定量测量是许多实验所需的终结点。然而，细胞外酸化的总速率是两个组件的总和:呼吸酸化， 以 CO 2（其水合物至H 2 CO 3，然后离解HCO 3 - + H +）的形式，和糖酵解酸化，在形式乳酸- + H +。 CO 2的总胞外酸化的贡献，直到最近被认为是可以忽略不计在这里使用的测量平台，XF24分析仪7。然而，很明显在该CO 2可以是一个主要贡献者细胞外酸化4-5多个其他系统。多篇论文承认这个骗子tribution，但不要试图二氧化碳直接定量衍生酸3,8,9。我们最近证明了定量的 CO 2的生产是细胞外酸化在这个系统6一个显著源。此外，虽然有生成二氧化碳从葡萄糖分解代谢的多个代谢途径，那些在三羧酸循环进行的矩阵脱氢酶是压倒性的贡献者和所有其他源产生的二氧化碳都在实验误差6的数额。 未经校正的CO 2的产生 ，细胞外酸化因此是糖酵解速率的暧昧指示器和不能定量使用。我们以前的出版物突出了几个实例，其中呼吸的 CO 2包括了大部分的总酸化信号的，即使是在一般认为主要使用糖酵解6个细胞 。此外，该呼吸的 CO 2贡献总酸化常见的代谢分析实验过程中变化很大，这表明在实验的不同部分的糖酵解速率的正确比较需要校正的 CO 2。 为了测量用细胞外酸化的速率细胞的糖酵解速率，有必要pH值的变化转换为所产生的变化总的 H +，并减去所造成的 CO 2的胞外酸化柠檬酸循环的操作过程中释放出来。在这里，我们描述了用于测量细胞外的质子产率（从pH值和细胞外的变化分析培养基的校准缓冲力）和CO 2的生产（从外变化O 2浓度）一个简单的方法，并展示如何计算糖酵解率使用这些测量。 这种方法的力量<html>通过使用它来正确地计算由乳酸产生所限定的糖酵解速率ENS细胞外酸化测量的效用。未经校正的呼吸的 CO 2（或直接测量乳酸），这是不可能，以确定是否和在何种程度上总酸化率反映糖酵解速率，混杂的使用总细胞外酸化作为直接测量乳酸产生的实验解释。 演算 二氧化碳和乳酸是，在实验误差内，仅有的两个贡献者细胞外产酸，是根据实验成肌细胞6。因此，总的细胞外酸化（PPR，质子产率）​​的速率可被定义为: PPR TOT = PPR RESP + PPR GLYC 公式1 。_content"&gt;其中TOT =总; RESP =呼吸; GLYC =糖酵解糖酵解PPR这样的: PPR GLYC = PPR TOT - PPR RESP 公式（2） 这里， PPR TOT = ECAR TOT / BP 公式3 其中ECAR =细胞外酸化率（MPH /分钟），和BP =缓冲功率（MPH /皮摩尔的 H +在7微升），而 PPR RESP =（10 pH值-PK 1 /（1 + 10 pH值-PK 1））（最大值H + / O 2）（OCR TOT - OCR 转/ MYX） 公式4 其中K 1 = CO 2的水化和分解到HCO 3组合平衡常数- + H +;最大H + / O 2 =日ËCO 2来源的酸化为特定的代谢转化，如葡萄糖6完全氧化; OCR =氧消耗速率（皮摩尔O 2 /分钟），和OCR 腐/ MYX =不可线粒体OCR。 方程4株的线粒体的OCR减去任何非线粒体的OCR（定义为OCR是 ​​抗线粒体呼吸毒药鱼藤酮和myxothiazol），占最大的 H +每消耗的O 2的每个基片（最大的 H + / O 2产生 ）（见第6），以及二氧化碳从而产生的 H +在实验温度和pH（10 的pH的pK 1 /（1 + 10 的pH的pK 1的比例），对于葡萄糖的充分氧化，线粒体氧消费率（OCR）正好等于CO 2的产率。在细胞外通量测量的密闭测定体积，CO 2的产生D由呼吸仍被困在试验中。大部分的捕获的 CO 2水合至H 2 CO 3，然后离解HCO 3 - + H +。一小部分仍溶解但不能水合，而另一小部分是水合的但不是离解的，如决定热力学由CO 2水合和离解的组合平衡常数来HCO 3 - + H +在实验温度（37℃）和pH值（〜7.4）。 因此，完整的公式计算PPR 克减去PPR RESP从PPR TOT是: PPR GLYC = ECAR 托特 / BP - （10 的pH的pK 1 /（1 + 10 的pH的pK 1））（最大的 H + / O 2）（OCR托特 - OCR 腐/ MYX） 等式5 一世n这个方式，呼吸和糖酵解，以及它们相关的ATP生产速率的速率，可以定量从简单的测量（耗氧量，外酸化，缓冲能力），以及其他所需的值进口或计算（H + / O 2的决定的P / O和平衡常数K 1）6。这里描述的实验进一步阐述的标准技术，使用细胞外通量分析仪如海马XF24 10,11;其他外通量测量格式 （例如，XFê96，或XFP），低于所有卷应适当缩放。 分析培养基的缓冲功率可以通过构造一个标准曲线的或者直接在细胞外通量平台或单独使用校准pH电极进行测定。在这里，三个选项供外通量测定法中测量缓冲中给出，其中包括使用所有injecti在细胞外通量分析仪无细胞样品井，或者仅使用在井含细胞（第1部分），或通过使用外部pH测量（部分2）在最后一次注射端口的端口。见所附的电子表格例如数据的全部计算。 要使用外流量仪表的pH值检测能力衡量缓冲能力，这是最安全的使用无细胞井，以尽量减少信号变化。然而，误差范围内，无细胞和进行该测量时井含细胞（数据未示出）之间无显着差异存在。注:在步骤1.7中描述的变异进行占通过加入化合物或通过细胞的存在赋予缓冲任何可能的变化，与信号噪声较大的缺点的优点。然而，如上所述，被发现在无细胞之间设计在表1所示的计算出的缓冲功率无显著差异和后实验设计在表2这里所述的实验条件下进行。 另外，过小ΔpH范围（&lt;0.4单位;实验最好限定为0.2单位）中，线性斜率获得通过绘制ΔMPH /皮摩尔H +充分近似于ΔpH和[H +]之间的对数关系。因此该标准曲线的斜率表示了测定培养基的被测的pH /纳摩尔H +缓冲功率在7微升，或在7微升MPH /皮摩尔H +。我们建议增加介质缓冲功率或降低细胞密度超过在测量时间0.2 pH单位变化的样品。测量时间，也可以减少，但是这可能缩短稳态酸化速率和将误差引入的速率计算。

<table><tbody><tr><td>Pherastar FS</td><td>BMG</td><td>n/a</td><td>microplate reader</td></tr><tr><td>Seahorse XF-24</td><td>Seahorse Bioscience</td><td>n/a</td><td>extracellular flux instrument</td></tr><tr><td>Seahorse XF assay plate</td><td>Seahorse Bioscience</td><td>V7-PS</td><td>consumable</td></tr><tr><td>XF Calibrant</td><td>Seahorse Bioscience</td><td>100840-000</td><td>solution</td></tr><tr><td>HCl standard</td><td>Sigma</td><td>38280</td><td>chemical</td></tr><tr><td>oligomycin</td><td>Sigma</td><td>O4876</td><td>chemical</td></tr><tr><td>FCCP</td><td>Sigma</td><td>C2920</td><td>chemical</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R8875</td><td>chemical</td></tr><tr><td>Myxothiazol</td><td>Sigma</td><td>T5580</td><td>chemical</td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td>medium component</td></tr><tr><td>FBS</td><td>Corning</td><td>35-010-CV</td><td>medium component</td></tr><tr><td>penicillin/streptomycin</td><td>Corning</td><td>30-002-CI</td><td>medium component</td></tr><tr><td>carbonic anhydrase</td><td>Sigma</td><td>C2624</td><td>chemical</td></tr><tr><td>96-well assay plate</td><td>Corning</td><td>CLS3991</td><td>consumable</td></tr><tr><td>NAD&lt;sup&gt;+&lt;/sup&gt;</td><td>Sigma</td><td>N7004</td><td>chemical</td></tr><tr><td>LDH</td><td>Sigma</td><td>L1254</td><td>chemical</td></tr></tbody></table>

measurement and analysis of extracellular acid production to determine glycolytic rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

<html> 图2示出的原始数据的典型实验。从点对点记录既OCR和pH值（阴影竖条）的最后10个测量点被用于计算。最初担心，每个检测周期的平均值（中间点测量）将不是一个精确的计算提供的速率足够高的分辨率，特别是似乎有口除了和稳态酸化率之间存在轻微的滞后，并没有证实，因为这似乎没有显著到计算误差贡献（未示出）。另外，如果正确的缓冲能力是实验设置期间输入，PPR可直接从仪器收集数据的读出，通过显示在仪器的软件或在PC兼容的格式可用作为数据输出设置中的一种在PPR输出读取。 ftp_upload / 53464 / 53464fig2.jpg"/&gt; 图中O 2和H + 2代表外通量的痕迹 。 OCR和pH值跟踪的示例实验在表5中，含有10mM葡萄糖测定开始。端口D具有不同的HCl浓度为缓冲功率的校准（在这些平均迹线未示出）。从以前的出版物6数据 。每个点代表平均每组8生物学重复±SEM。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53464/53464fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 的代表性结果数据分析 使用表 6中所示，并提供作为附件的电子表格中，从各个孔的数据值可以与黄色头标中表示的列中输入。所有六个列<html>右，从这些项来计算。 表 6中的例子示出PPR RESP和使用ECAR和OCR数据从单个孔用于加入或不加入葡萄糖的天然条件，前端口A加成寡PPR GLYC的计算。在每个生物制剂技术重复通常平均以得到输出的单​​一值在最后四列，然后从不同的生物制剂的数据进行平均以适当的血压错误统计和这四个值的传播。 <img alt="表6" src="/files/ftp_upload/53464/53464table6.jpg" /> 表6.计算呼吸和糖酵解酸化。列在黄色为首指示值从计算输入（例如，BP，最大的 H + / O 2），或从数据收集（例如，ECAR 托特 ，OCR）。<html> PS://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53464/53464table6.jpg"目标="_空白"&gt;请点击这里查看此表的放大版|。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53464/53464_Table_6.xlsx">请点击此处下载此表为Excel电子表格。</a> 糖酵解和呼吸的贡献PPR修正后 图3示出计算如表6糖酵解和呼吸酸化以下FCCP加成（端口B），原产率，以下寡加成（端口A）比率，和价格数据的图形输出。这些数据清楚地说明了如何呼吸和糖酵解酸化变化选择基板的比例（相对于血糖控制（CTL），以无添加），并与线粒体状态（本机的功能与药理功能改变）。 "&gt; <img alt="图3" src="/files/ftp_upload/53464/53464fig3.jpg" /> 从糖酵解和呼吸源图3.质子产率（PPR）。 PPR从呼吸（开口部分），并使用等式5添加了葡萄糖（3左侧柱）或不添加葡萄糖（3右柱）计算C2C12细胞的糖酵解（填充的部分）。从6数据 。所有的数据表示平均的n±标准差= 8生物学重复。

Watch this Scientific Journal Video about Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate at JoVE.com

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

measurement-analysis-extracellular-acid-production-to-determine

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

25.0K 次观看.  Touro University California College of Pharmacy. Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

视频: Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

科研

癌症研究

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

免疫与感染

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

人类自然杀手细胞代谢分析

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

神经科学

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

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 ...

生物学

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

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 ...

测量外产酸和分析，以确定糖酵解率

摘要

探索更多视频

此视频中的章节

原发性白血病细胞代谢谱的评价

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

人类自然杀手细胞代谢分析

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量

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

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

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

测量外产酸和分析，以确定糖酵解率

摘要

探索更多视频

此视频中的章节

原发性白血病细胞代谢谱的评价

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

人类自然杀手细胞代谢分析

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

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

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

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

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量