Name: measuring trans-plasma membrane electron transport by c2c12 myotubes
Uploaded: 2018-05-04T15:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

我们要感谢托马斯贝尔, 林恩 Mattathil, 马克 Mannino, 和 Neej 的技术支持。这项工作得到美国公共卫生服务奖 R15DK102122 从国家糖尿病和消化和肾脏疾病研究所 (NIDDK) 的支持乔纳森费舍尔。手稿内容完全是作者的责任, 不一定代表 NIDDK 或国立卫生研究院的官方意见。

注意: 有关关键步骤的示意图概述, 请参见图 1 。
1. WST-1 还原试验
<ol><li>使用标准的细胞培养过程在96井板中利用 a F 来生长和区分 C2C12 黏附细胞.7<ol>
<li>使用由 Dulbecco 修饰鹰培养基 (DMEM) 组成的分化培养基, 辅以2% 匹马血清、100毫升青霉素和0.1 毫克/毫升链霉素。孵化细胞在37°c 与 5% CO2。</li>
		<li>当监测抗坏血酸参与 tPMET, 补充分化培养基与100µM 抗坏血酸。允许细胞在分化培养基中孵育24-48 小时。</li>
	</ol>
</li>
	<li>准备库存 WST-1 和 PMS 解决方案。<ol>
<li>为了使10毫米的 WST-1, 溶解0.033 克 WST-1 (FW): 651.34 克/摩尔) 在5毫升磷酸盐缓冲盐水 (PBS)。存储在4摄氏度。</li>
		<li>要制作5毫米的 pms 库存, 可将0.0023 克 pms (固件: 306.34 克/摩尔) 溶解在1.5 毫升的去离子 H2o (蝶和 2 o).储存在-20 摄氏度和保护免受光照。</li>
	</ol>
</li>
	<li>添加0.0108 克葡萄糖的最终浓度为5毫米至11.4 毫升的 PBS 或 HEPES 缓冲生理盐水 (哈佛商学院; 20 毫米 HEPES 钠盐, 140 毫米氯化钠, 5 毫米氯化钾, 2.5 毫米 MgSO4, 1 毫米 CaCl2)。添加480µL 10 毫米 WST-1 为最终浓度为400µM 和48µL 5 毫米 PMS 的最后浓度20µM。</li>
	<li>当监测抗坏血酸参与 tPMET, 把溶液分成两个6毫升整除数。对一个整除, 添加6µL 蝶和 2 O 和另一个整除添加6µL 2 kU/毫升 AO 为最终浓度 2 U/毫升.</li>
	<li>当监测超氧化物参与 tPMET, 将解决方案分为两个6毫升整除数。对于一个整除, 添加55µL KPO4缓冲区和另一个整除添加55µL 6.5 kU/毫升 SOD, 以最终浓度为 60 U/毫升。</li>
	<li>用 PBS 冲洗细胞。吸入介质并添加150µL PBS。然后吸入 PBS 注: 化验是用电镀的, 附着的细胞做的。因此, 在洗涤过程中没有离心或胰蛋白酶的使用。</li>
	<li>添加100µL WST-1 溶液 (-) sod 或 (-) ao 到列 1-6, 并添加100µL WST-1 溶液 (+) SOD 或 (+) AO 到7-12 列在96井板块。使用行 G 和 H 作为背景控件 (即, 在没有单元格的井中单独监测试剂中吸光度的变化)。</li>
	<li>用分光光度计每10分钟测量1小时的吸光度值 438 nm。</li>
	<li>读完后, 用150µL 的 PBS 冲洗介质, 洗净每一个井。然后吸入 PBS</li>
	<li>计算每个井吸光度的变化, 方法是从任何时间点的吸光度中减去井的初始吸光度。更正背景井 (即) 中观察到的吸光度变化 (如果有) 的变化, 而不使用检测溶液, 但没有细胞的油井。</li>
	<li>为了进行分析, 将吸光度数据正常化为60分钟的控制测量或用 PBS 或哈佛商学院冲洗水井, 然后执行二辛可宁酸 (可评估) 蛋白质测定。</li>
	<li>添加2毫克/毫升牛血清白蛋白 (BSA) 标准 (范围从 0.5-2 µL 为标准曲线, 取决于细胞的汇合程度) 到空井 (G 行和 H), 然后添加到所有水井的可测性试剂。</li>
	<li>通过 nmol 每µg 蛋白质的 WST-1 减少来量化数据。使用37毫米-1 cm-1 22作为消光系数为减少的 WST-1 在438毫微米。</li>
</ol>2. DPIP 还原试验
<ol><li>生长和区分 C2C12 黏附细胞与步骤1.1 相同的步骤。</li>
	<li>准备库存10毫米 DPIP 溶液如下。溶出0.029 克 DPIP (FW: 290.08 克/摩尔) 在10毫升的蝶和 2 o. 通过用分光光度计测定 600 nm 中的吸光度, 确定 DPIP 的浓度.使用1毫米-1 cm-1 23作为消光系数为减少的 DPIP 在600毫微米。存储在4摄氏度。</li>
	<li>添加0.0108 克葡萄糖到11.880 毫升的 PBS, 最终浓度为5毫米。添加120µL 10 毫米 DPIP 的最后浓度为100µM。</li>
	<li>当监测抗坏血酸参与 tPMET, 把溶液分成两个6毫升整除数。对一个整除, 添加6µL 蝶和 2 O 和另一个整除添加6µL 2 kU/毫升 AO 为最终浓度 2 U/毫升.</li>
	<li>当监测超氧化物参与 tPMET, 将解决方案分为两个6毫升整除数。对于一个整除, 添加55µL KPO4缓冲区和另一个整除添加55µL 6.5 kU/毫升 SOD, 以最终浓度为 60 U/毫升。</li>
	<li>在150µL 的 PBS 中吸入培养基和洗涤细胞。吸入 PBS 并添加100µL DPIP 溶液 (-) sod 或 (-) ao 到1-6 列, 并添加100µL DPIP 溶液 (+) SOD 或 (+) AO 到7-12 列在96井板。使用 G 和 H 行作为后台控制 (即), 以监测在没有细胞的油井中单独的试剂中吸收的变化。</li>
	<li>用分光光度计每10分钟测量 600 nm 的吸光度, 1 h. 将相对于该控件的吸光度的变化量化为60分钟, 类似于步骤 1.9-1.13。</li>
</ol>3. 降低电子受体是否为 AO 或 SOD 基板的测定
<ol><li>到5.436 毫升的 PBS 或哈佛商学院, 添加240µL 10 毫米 WST-1 为最终浓度400µM 和24µL 5 毫米 PMS 的最后浓度20µM。<ol>
<li>对于 DPIP, 添加60µL 10 毫米 DPIP, 最终集中100µM, 到5.940 毫升的 PBS 或哈佛商学院。</li>
	</ol>
</li>
	<li>在没有细胞的情况下, 在一个平底96井板中添加100µL 溶液, 并测量分光光度计在 438 nm, WST-1, 或 600 nm, DPIP。</li>
	<li>添加1µL 10 毫米抗坏血酸到一半的水井, 最后浓度为100微米。监测吸光度直到稳定。</li>
	<li>稳定后, 添加1µL 200 u/毫升 AO 的最终浓度为 2 u/毫升的每一个井或添加1µL 6 kU/毫升 SOD 的最后浓度 60 U/毫升的最终集中到每一个井和监测吸光度为 1 h。</li>
</ol>

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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=Myricetin+induces+pancreatic+cancer+cell+death+via+the+induction+of+apoptosis+and+inhibition+of+the+phosphatidylinositol+3-kinase+(PI3K)+signaling+pathway.">Myricetin induces pancreatic cancer cell death via the induction of apoptosis and inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway.</a> Cancer Letters. 308 (2), 181-188 (2011).</li><li>Altundag, E., 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=Quercetin-induced+cell+death+in+human+papillary+thyroid+cancer+(B-CPAP)+cells.">Quercetin-induced cell death in human papillary thyroid cancer (B-CPAP) cells.</a> Journal of thyroid research. 2016 (8), (2015).</li><li>Ukeda, H., Kawana, D., Maeda, S., Sawamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Assay+for+Superoxide+Dismutase+Based+on+the+Reduction+of+Highly+Water-soluble+Tetrazolium+Salts+by+Xanthine-Xanthine+Oxidase.">Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction of Highly Water-soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase.</a> Biosci Biotechnol Biochem. 63 (3), 485-488 (1999).</li><li>Halaka, F. G., Babcock, G. T., Dye, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+5-methylphenazinium+methyl+sulfate.+Reaction+of+the+oxidized+form+with+NADH+and+of+the+reduced+form+with+oxygen.">Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen.</a> J Biol Chem. 257 (3), 1458-1461 (1982).</li><li>Maghzal, G. J., Krause, K. H., Stocker, R., Jaquet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+species+derived+from+the+family+of+NOX+NADPH+oxidases.">Detection of reactive oxygen species derived from the family of NOX NADPH oxidases.</a> Free Radic Biol Med. 53 (10), 1903-1918 (2012).</li></ol>

作者没有什么可透露的。

我们提出了两种方法, 利用细胞外电子受体, WST-1 和 DPIP, 分光光度法检测 tPMET C2C12 myotubes。随着标准培养过程中细胞系的生长和分光光度计板读取器的增加, 在简单的微板块实验中, 可以用这些电子受体监测 tPMET。WST-1 的还原是从良好到良好的在一个试验中重现, 但有日常的变化。以 PBS 为缓冲器的日日变化系数 (cv) 为 0.18, 以哈佛商学院为缓冲区的 cv 为0.23。
从以前的文献使用 ...

纯化的等离子膜降低电子受体的能力使人认为等离子膜具有固有的氧化还原容量1。以前在真菌、植物和动物中看到过, tPMET 是多个有机体共有的过程2,3,4,5。具体地说, 这一过程已在酿酒酵母、胡萝卜细胞、红细胞、淋巴细胞、骨肉瘤、黑色素瘤、巨噬细胞、骨骼肌和中性粒细胞2、3、4,5,6,7. 在通过等离子膜传输电子以减少细胞外氧化剂的过程中, tPMET 涉及许多细胞功能, 包括: 细胞生长5,8, 细胞生存能力9, 铁新陈代谢10, 细胞信号11,12,13, 和保护从活性氧种类12,14,15。由于 tPMET 在许多细胞功能中的参与, tPMET 的失衡已经被推测为某些严重的健康状况的发展做出贡献, 包括癌症16、心血管疾病17和新陈代谢综合症18。
有多种方法来监测电子在等离子膜上的传输, 但最广泛使用的技术是通过比色测定来评估细胞外电子受体的减少。通常使用的细胞外电子受体是四氮唑盐、DPIP、FeCN 和 ferricytochromec19, 20.最常用的四氮唑盐是第二代盐, 称为WST-119。与第一代四氮唑盐相比, 这种化合物在比色测定中更容易使用, 因为两个磺酸基团增加了其水溶性21。WST-1 与中间电子受体 1-甲氧基吩 methosulfate (mPMS) 相结合, 在两个单电子转移事件中减少。此还原将 WST-1 的弱色氧化形式更改为更强烈的黄色 formazan20,22。WST-1 有一个高摩尔消光系数 37 x 103 M-1cm-1, 导致高检测灵敏度21,22。DPIP 也被用作细胞外电子受体来监测 tPMET。已表明, DPIP 可以减少 extracellularly 由 tPMET 不支持中间电子受体23,24。由于缺少中间电子受体, DPIP 可以直接从等离子膜中提取电子, 而不像 WST-124。类似于 DPIP, FeCN 已被证明是减少 extracellularly 盐 tPMET 不支持中间电子受体19,24。与 WST-1 和 DPIP 不同, FeCN 具有低摩尔消光系数, 导致检测灵敏度降低9。另一个常用的细胞外电子受体监测 tPMET 是 ferricytochrome c. 类似 WST-1, ferricytochrome c 减少增加使用中间电子受体, mPMS22。与 WST-1 不同的是, ferricytochrome c 方法由于高背景和低摩尔消光系数22而不太敏感。
本文提出了一种通过分光光度法进行 tPMET 的实时分析方法。该方法利用细胞外电子受体 WST-1 和 DPIP, 因为它们都具有较高的摩尔消光系数, 而与其他常用的细胞外电子受体如 ferricytochrome c 相比, 它们的成本更低。我们使用吩 methosulfate (pms), 而不是 mPMS 他们有类似的化学成分和 PMS 是远远低于昂贵。mPMS 是光化学法稳定, 这是一个重要的特点, 商业套件, 需要长期的货架期。然而, 我们使 PMS 新的每一个化验, 所以稳定性不应该是一个问题。我们还提出一种方法来评估可能的酶相互作用 (参见图 1) 之间的细胞外电子受体和酶, 可用于进一步表征的过程 tPMET。具体地说, 酶的 AO 和 SOD 可以用来确定哪一部分的 tPMET 是由于抗坏血酸转运或胞外超氧化物释放, 两种常用的方法, 电子运输横跨等离子膜。

<table><tbody><tr><td>C2C12 myoblasts</td><td>American Type Culture Collection&amp;nbsp;</td><td>CRL-1772</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium - low glucose</td><td>Sigma</td><td>D6046</td><td></td></tr><tr><td>Fetal Plex animal serum complex</td><td>Gemini Bio-Products&amp;nbsp;</td><td>100-602</td><td></td></tr><tr><td>penicillin-streptomycin</td><td>Sigma</td><td>516106</td><td></td></tr><tr><td>horse serum</td><td>Gibco Technologies</td><td>16050-130</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>15 cm culture dishes</td><td>TPP</td><td>93150</td><td></td></tr><tr><td>96 well culture plates</td><td>TPP</td><td>92096</td><td></td></tr><tr><td>2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1)</td><td>Accela ChemBio&amp;nbsp; Inc</td><td>SY016315</td><td></td></tr><tr><td>phenazine methosulfate&amp;nbsp;</td><td>Sigma</td><td>P9625</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>ascorbate oxidase&amp;nbsp;</td><td>Sigma</td><td>A0157</td><td></td></tr><tr><td>superoxide dismutase&amp;nbsp;</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>2,6-dichloroindophenol sodium salt&amp;nbsp;</td><td>ICN Biomedicals</td><td>215011825</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>HEPES sodium salt</td><td>Sigma</td><td>H3784</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>potassium chloride</td><td>Fisher Scientific&amp;nbsp;</td><td>BP366</td><td></td></tr><tr><td>magnesium sulfate heptahydrate</td><td>Sigma</td><td>M5921</td><td></td></tr><tr><td>calcium chloride dihydrate</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>potassium phosphate</td><td>Fisher Scientific&amp;nbsp;</td><td>BP363</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Powerwave X-I spectrophotometer</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr><tr><td>Spectronic Genesys 5 Spectrophotometer</td><td>Thermo Scientific</td><td>336001</td><td></td></tr><tr><td>PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterile</td><td>MidSci</td><td>781602</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma</td><td>220299</td><td></td></tr><tr><td>Iron (II) sulfate heptahydrate</td><td>Sigma</td><td>215422</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma</td><td>H9636</td><td></td></tr><tr><td>xanthine oxidase</td><td>Sigma</td><td>X4500</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>R Studio</td><td>Rstudio</td><td></td><td>https://www.rstudio.com/products/rstudio/</td></tr><tr><td>KC4</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr></tbody></table>

measuring trans-plasma membrane electron transport by c2c12 myotubes

反式等离子体膜电子传输 (tPMET) 在细胞内的还原应力保护和保护免受细胞外氧化剂的破坏中起着重要作用。这种从胞内还原剂向细胞外氧化剂输送电子的过程没有很好的定义。在这里, 我们提出了 C2C12 myotubes 的分光光度检测, 以监测 tPMET 利用细胞外电子受体: 水溶性四氮唑 salt-1 (WST-1) 和 26-dichlorophenolindophenol (DPIP 或 DCIP)。通过减少这些电子受体, 我们能够在实时分析中监控这个过程。随着抗坏血酸氧化酶 (AO) 和超氧化物歧化酶 (SOD) 的加入, 我们可以确定 tPMET 的哪一部分是由抗坏血酸出口或超氧化物产生的。当 WST-1 在低背景下产生稳定结果的同时, 在添加 AO 和 SOD 后, DPIP 能够重新氧化, 并通过分光光度分析证明。该方法显示了一种实时、多井、快速分光光度法, 其优点优于其它用于监测 tPMET 的方法, 如氰化钾 (FeCN) 和 ferricytochrome c 还原法。

使用 RStudio 统计软件25进行重复测量, 对方差分析进行了统计。示例大小在图图例中显示。
为了监测 tPMET, C2C12 myotubes 与细胞外电子受体、WST-1 和 DPIP 一起使用。AO 用于确定 WST-1 和 DPIP 减少的部分是由于抗坏血酸流出和 SOD 被用来确定 WST-1 减少的部分是由于胞外超氧化物释放。如图 2所?...

Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

C2C12 Myotubes 测量反式等离子体膜电子传输

本协议的目标是通过细胞外电子受体 spectrophotometrically 监测反式等离子体膜电子传输, 并分析这些细胞外电子受体可能发生的酶相互作用。

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

measuring-trans-plasma-membrane-electron-transport-by-c2c12-myotubes

Research

JoVE Journal

Biochemistry

6.9K 次观看.  Saint Louis University. 本协议的目标是通过细胞外电子受体 spectrophotometrically 监测反式等离子体膜电子传输, 并分析这些细胞外电子受体可能发生的酶相互作用。

视频: Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

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

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

医学

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

测量来自母猪后代的先前冷冻心脏组织中的线粒体电子转移复合物:评估运动诱导的线粒体生物能量变化的模型

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

生物化学

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

线粒体内膜对Na+ 的敏感性揭示了部分分割的功能性辅酶Q库

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

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

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

在C2C12肌细胞造型强直性肌营养不良1

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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.

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 ...