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>

<ol>
	<li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochem. J. 435 (2), 297-312 (2011).</li><li>Gerencser, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+microplate-based+respirometry+with+correction+for+oxygen+diffusion.">Quantitative microplate-based respirometry with correction for oxygen diffusion.</a> Anal. Chem. 81 (16), 6868-6878 (2009).</li><li>Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+interpretation+of+microplate-based+oxygen+consumption+and+pH+data.">Analysis and interpretation of microplate-based oxygen consumption and pH data.</a> Meth. Enzymol. 547, 309-354 (2014).</li><li>Renner, K., Jansen-D&#252;rr, P., Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+oxygen+kinetics+of+cellular+respiration+and+linear+oxygen+dependence+of+antimycin+A+inhibited+oxygen+consumption.">Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption.</a> Mol. Biol. Rep. 29 (1-2), 83-87 (2002).</li><li>Helmlinger, G., Sckell, A., Dellian, M., Forbes, N. S., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+production+in+glycolysis-impaired+tumors+provides+new+insights+into+tumor+metabolism.">Acid production in glycolysis-impaired tumors provides new insights into tumor metabolism.</a> Clin. Cancer Res. 8 (4), 1284-1291 (2002).</li><li>Mookerjee, S. A., Goncalves, R. L. S., Gerencser, A. G., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contributions+of+respiration+and+glycolysis+to+extracellular+acid+production.">The contributions of respiration and glycolysis to extracellular acid production.</a> Biochim. Biophys. Acta. 1847, 171-181 (2015).</li><li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+metabolic+analysis+reveals+a+close+link+between+attenuated+mitochondrial+bioenergetic+function+and+enhanced+glycolysis+dependency+in+human+tumor+cells.">Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.</a> Am. J. Physiol. 292 (1), C125-C136 (2006).</li><li>Nadanaciva, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+drug-induced+mitochondrial+dysfunction+via+altered+cellular+respiration+and+acidification+measured+in+a+96-well+platform.">Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform.</a> J. Bioenerg. Biomembr. 44 (4), 421-437 (2012).</li><li>Pelletier, M., Billingham, L. K., Ramaswamy, M., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+flux+analysis+to+monitor+glycolytic+rates+and+mitochondrial+oxygen+consumption.">Extracellular flux analysis to monitor glycolytic rates and mitochondrial oxygen consumption.</a> Meth. Enzymol. 542, 125-149 (2014).</li><li>Nicholls, D. G., Darley-Usmar, V. M., Wu, M., Jensen, P. B., Rogers, G. W., Ferrick, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+profile+experiment+using+C2C12+myoblast+cells.">Bioenergetic profile experiment using C2C12 myoblast cells.</a> J. Vis. Exp. (46), 2511 (2010).</li><li>Rogers, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PLoS ONE. 6 (7), e21746 (2011).</li><li>Seahorse Biosciences. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> F24 Extracellular Flux Analyzer and Prep Station Installation and Operation Manual. 1.7, (2010).</li><li>Blau, H., Chiu, C., Webster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+activation+of+human+nuclear+genes+in+human+heterocaryons.">Cytoplasmic activation of human nuclear genes in human heterocaryons.</a> Cell. 32, 1171-1180 (1983).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<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+</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://www.jove.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：//www.jove.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://www.jove.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

Research

JoVE Journal

Biology

24.9K 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

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

科研

生物学

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

细胞外离子流的使用离子选择性自引用的微电极技术测量

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

从放射性标记的底物在二氧化碳生成的测量<em&gt;果蝇</em

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human ...

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 ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

细胞调理介质细胞外囊泡的流式细胞学分析

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

细胞外囊泡的量化和尺寸测定纳米粒子跟踪分析

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

使用实时显微镜和开源软件对 阿斯珀吉卢斯尼杜兰 生长速度的定量分析

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...