Name: assessing energy substrate oxidation in vitro with 14co2 trapping
Uploaded: 2022-03-23T13:30:00
Description: Watch this Scientific Journal Video about Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping at JoVE.com

这项工作得到了NIH拨款R01 AR060456（FL）的部分支持。我们感谢Michael Robinson博士和Elizabeth Krizman（费城儿童医院）对闪烁计数器的慷慨帮助。

放射性物质（RAM）的使用需要事先得到每个机构的指定安全委员会的批准。该协议中使用的RAM已获得宾夕法尼亚大学环境健康与辐射安全（EHRS）的批准。使用动物需要事先获得家庭机构的机构动物护理和使用委员会（IACUC）的批准。以下研究由费城儿童医院的IACUC批准。
1. 14种C标记底物的储备溶液的制备
<ol><li>通过将11gBSA和33.1mL Dulbecco的磷酸盐缓冲盐水...

<ol>
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Effects of fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes and starvation, on the uptake and metabolic fate of glucose in rat heart and diaphragm muscles.</a> Biochemical Journal. 93 (3), 652-665 (1964).</li><li>Taegtmeyer, H., Hems, R., Krebs, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+energy-providing+substrates+in+the+isolated+working+rat+heart.">Utilization of energy-providing substrates in the isolated working rat heart.</a> Biochemical Journal. 186 (3), 701-711 (1980).</li><li>Cha, B. 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=Impaired+fatty+acid+metabolism+in+type+2+diabetic+skeletal+muscle+cells+is+reversed+by+PPARgamma+agonists.">Impaired fatty acid metabolism in type 2 diabetic skeletal muscle cells is reversed by PPARgamma agonists.</a> American Journal of Physiology. Endocrinology and Metabolism. 289 (1), 151-159 (2005).</li><li>Lee, W. C., Guntur, A. R., Long, F., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+the+osteoblast:+implications+for+osteoporosis.">Energy metabolism of the osteoblast: implications for osteoporosis.</a> Endocrine Reviews. 38 (3), 255-266 (2017).</li><li>Li, B., 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=Both+aerobic+glycolysis+and+mitochondrial+respiration+are+required+for+osteoclast+differentiation.">Both aerobic glycolysis and mitochondrial respiration are required for osteoclast differentiation.</a> FASEB Journal. 34 (8), 11058-11067 (2020).</li><li>Lee, W. C., Ji, X., Nissim, I., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malic+enzyme+couples+mitochondria+with+aerobic+glycolysis+in+osteoblasts.">Malic enzyme couples mitochondria with aerobic glycolysis in osteoblasts.</a> Cell Reports. 32 (10), 108108 (2020).</li><li>Kim, S. P., 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=Fatty+acid+oxidation+by+the+osteoblast+is+required+for+normal+bone+acquisition+in+a+sex-+and+diet-dependent+manner.">Fatty acid oxidation by the osteoblast is required for normal bone acquisition in a sex- and diet-dependent manner.</a> JCI Insight. 2 (16), 92704 (2017).</li><li>Yu, Y., 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=Glutamine+metabolism+regulates+proliferation+and+lineage+allocation+in+skeletal+stem+cells.">Glutamine metabolism regulates proliferation and lineage allocation in skeletal stem cells.</a> Cell Metabolism. 29 (4), 966-978 (2019).</li><li>Karner, C. M., Esen, E., Okunade, A. L., Patterson, B. W., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutamine+catabolism+mediates+bone+anabolism+in+response+to+WNT+signaling.">Increased glutamine catabolism mediates bone anabolism in response to WNT signaling.</a> The Journal of Clinical Investigation. 125 (2), 551-562 (2015).</li><li>Takeshita, S., Kaji, K., Kudo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+the+new+osteoclast+progenitor+with+macrophage+phenotypes+being+able+to+differentiate+into+mature+osteoclasts.">Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts.</a> Journal of Bone and Mineral Research. 15 (8), 1477-1488 (2000).</li><li>Itoh, Y., 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=Dichloroacetate+effects+on+glucose+and+lactate+oxidation+by+neurons+and+astroglia+in+vitro+and+on+glucose+utilization+by+brain+in+vivo.">Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro and on glucose utilization by brain in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (8), 4879-4884 (2003).</li><li>Oba, M., Baldwin, R. L. 4. t. h., Bequette, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+glucose,+glutamate,+and+glutamine+by+isolated+ovine+enterocytes+in+vitro+is+decreased+by+the+presence+of+other+metabolic+fuels.">Oxidation of glucose, glutamate, and glutamine by isolated ovine enterocytes in vitro is decreased by the presence of other metabolic fuels.</a> Journal of Animal Science. 82 (2), 479-486 (2004).</li><li>Esen, E., Lee, S. Y., Wice, B. M., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTH+promotes+bone+anabolism+by+stimulating+aerobic+glycolysis+via+IGF+signaling.">PTH promotes bone anabolism by stimulating aerobic glycolysis via IGF signaling.</a> Journal of Bone and Mineral Research. 30 (11), 1959-1968 (2015).</li><li>Huynh, F. K., Green, M. F., Koves, T. R., Hirschey, M. 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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=Comprehensive+profiling+of+plasma+fatty+acid+concentrations+in+young+healthy+Canadian+adults.">Comprehensive profiling of plasma fatty acid concentrations in young healthy Canadian adults.</a> PLoS One. 10 (2), 0116195 (2015).</li></ol>

该协议提供了一种易于使用的方法来确定主要能量基板的氧化速率。它是其他方案的更简单的替代方案，这些方案使用包含中央孔并盖有橡胶塞14，15，16的烧瓶。尽管这里的示例研究是用细胞培养进行的，但该方法可以很容易地适用于含有完整线粒体的组织外植体或组织匀浆，如前所述17。
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真核生物中的氧化磷酸化（OXPHOS）是营养物质在线粒体内部分解的过程，通过消耗氧气以ATP的形式释放化学能。 线粒体内部各种底物通过三羧酸（TCA）循环的分解代谢直接产生很少的ATP分子，而是通过还原电子载体烟酰胺腺嘌呤二核苷酸（NAD +）和黄素腺嘌呤二核苷酸（FAD +）来储存能量。然后，还原的载流子被位于线粒体内膜上的ETC氧化，以产生穿过膜的质子浓度梯度。质子最终通过ATP合酶沿着梯度流回线粒体基质中，产生ATP。OXPHOS是从能量基材生产ATP的最有效手段，通常在有氧环境中是优选的。以前，有氧糖酵解 - 在存在氧气的情况下从葡萄糖中产生乳酸盐 - 被认为是病理生理学的，通常是癌细胞的标志。越来越多的人发现，一些正常细胞类型使用有氧糖酵解的原因尚未完全破译。
代谢灵活性是细胞或生物体适应不断变化的能量需求和可用燃料来源的能力。例如，骨骼肌的能量需求主要由稳定状态下的OXPHOS满足，但在高强度运动中通过厌氧糖酵解满足1。随着运动持续时间的增加，葡萄糖和脂肪酸氧化对整体能量产生的贡献更大2.然而，基板的使用不仅取决于可用性，因为基底在氧化过程中具有拮抗性竞争。最值得注意的是，脂肪酸氧化已被证明可以抑制骨骼肌对葡萄糖的利用，这种现象称为Randle效应3。随后的研究4，5证明了互惠效应。此外，许多疾病与底物偏好的变化和细胞代谢不灵活性的发展有关。例如，与正常对照组相比，II型糖尿病患者的骨骼肌中的脂肪酸氧化减少6。疾病环境中的代谢变化是一个需要深入研究的主题，因为它们可能有助于发病机制。
骨骼细胞类型中的能量代谢相对较少，但近年来引起了人们的关注7.先前的研究表明，有氧糖酵解是颅变成骨细胞中的主要能量途径，而通过TCA循环的葡萄糖氧化在破骨细胞形成中起作用8，9。其他人已经提供了脂肪酸作为成骨细胞能量来源的证据10。谷氨酰胺分解代谢也被证明支持成骨细胞与祖细胞的分化11，12。然而，对各种骨骼细胞类型的底物利用的全面了解仍然缺乏。此外，细胞分化过程中细胞代谢的变化或对病理信号的反应预计将改变燃料底物的利用率。下面描述的是一种易于使用的方案，用于 在体外测定底物氧化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m filters</td>
			<td>Sigma-Aldrich</td>
			<td>SLGVM33RS</td>
			<td>Used to filter BSA solution</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>Dissociate cells from cell culture plates</td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>PR1MA</td>
			<td>PR MCT17 RB</td>
			<td>Used for reaction incubation</td>
		</tr>
		<tr>
			<td>10 cm plates</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>BD</td>
			<td>302995</td>
			<td>Used to flush marrow from long bones</td>
		</tr>
		<tr>
			<td>10% FBS</td>
			<td>Atlanta biologicals</td>
			<td>S11550</td>
			<td>For Cell culture medium preparation</td>
		</tr>
		<tr>
			<td>14C-Glucose</td>
			<td>PerkinElmer</td>
			<td>NEC042X050UC</td>
			<td>Used to make hot media</td>
		</tr>
		<tr>
			<td>14C-glutamine</td>
			<td>PerkinElmer</td>
			<td>NEC451050UC</td>
			<td>Used to make hot media</td>
		</tr>
		<tr>
			<td>14C-oleate</td>
			<td>PerkinElmer</td>
			<td>NEC317050UC</td>
			<td>Used to make hot media</td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>BD</td>
			<td>305120</td>
			<td>Used to flush marrow from long bones</td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>TPP</td>
			<td>92024</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainers</td>
			<td>MIDSCI</td>
			<td>70CELL</td>
			<td>Used to filter supernatant during cavarial digestion</td>
		</tr>
		<tr>
			<td>Acridine Orange/Propidium Iodide (AO/PI) dye</td>
			<td>Nexcelom Biosciences</td>
			<td>CS2-0106</td>
			<td>Stains live cells to determine seed density</td>
		</tr>
		<tr>
			<td>Bovine Serum Ablumin</td>
			<td>Proliant Biologicals</td>
			<td>68700</td>
			<td>Used for fatty acid conjugation</td>
		</tr>
		<tr>
			<td>Cellometer Auto 2000</td>
			<td>Nexcelom Biosciences</td>
			<td></td>
			<td>Determine the number of viable cells</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher</td>
			<td>Legend Micro 21R</td>
			<td>Used to pellet cells</td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td>Dissociate cells from tissue</td>
		</tr>
		<tr>
			<td>Custom MEM alpha</td>
			<td>GIBCO</td>
			<td>SKU: ME 18459P1</td>
			<td>Used to create custom hot media</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Used to dissolve and dilute reagents, and wash culture dishes</td>
		</tr>
		<tr>
			<td>Filter Paper</td>
			<td>Millipore-Sigma</td>
			<td>WHA1001090</td>
			<td>Traps CO2 with sodium hydroxide</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>g7528</td>
			<td>Used to make custom media</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td>Traps CO2 during cell culture</td>
		</tr>
		<tr>
			<td>L-carnitine</td>
			<td>Sigma-Aldrich</td>
			<td>C0283</td>
			<td>Supplemented for fatty acid oxidation</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>g3126</td>
			<td>Used to make custom media</td>
		</tr>
		<tr>
			<td>MEM alpha</td>
			<td>Thermo</td>
			<td>A10490</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Pecheney Plastic Packaging</td>
			<td>PM998</td>
			<td>Used to seal cell culture dishes</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td>Prevents contamination in cell culture</td>
		</tr>
		<tr>
			<td>Perchloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>244252</td>
			<td>Releases CO2 during metabolic assay</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>p5280</td>
			<td>Used to make custom media</td>
		</tr>
		<tr>
			<td>Scintillation Counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
			<td>Determines radioactivity from the filter paper</td>
		</tr>
		<tr>
			<td>Scintillation Fluid</td>
			<td>MP Biomedicals</td>
			<td>882453</td>
			<td>Absorb the energy emitted by RAMs and re-emit it as flashes of light</td>
		</tr>
		<tr>
			<td>Scintillation Vial</td>
			<td>Fisher Scientific</td>
			<td>03-337-1</td>
			<td>Reaction containers for scintillation fluid</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Balance buffer for medium</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>58045</td>
			<td>Traps CO2 during metaboilc assay</td>
		</tr>
		<tr>
			<td>Sodium oleate</td>
			<td>SANTA CRUZ</td>
			<td>SC-215879</td>
			<td>BSA conjugated fatty acid preparation</td>
		</tr>
		<tr>
			<td>Vaccum filtration 1000</td>
			<td>TPP</td>
			<td>99950</td>
			<td>Filter cMEM&#945;</td>
		</tr>
	</tbody>
</table>

assessing energy substrate oxidation in vitro with 14co2 trapping

线粒体是三羧酸（TCA）循环和电子传递链（ETC）的机器，其产生三磷酸腺苷（ATP）以维持能量稳态。葡萄糖，脂肪酸和氨基酸是大多数体细胞中促进线粒体呼吸的主要能量底物。有证据表明，不同的细胞类型可能对某些底物有明显的偏好。然而，骨架中各种细胞的底物利用尚未得到详细研究。此外，由于细胞代谢与生理和病理生理学变化相协调，对骨骼细胞底物依赖性的直接评估可能为骨骼疾病的发病机制提供重要的见解。
以下方案基于氧化磷酸化后底物分子释放二氧化碳的原理。通过使用含有放射性标记碳原子（14C）的底物，该方法为细胞培养物中的底物氧化速率提供了一种灵敏且易于使用的测定方法。一项以原代颅骨前成细胞与骨髓来源的巨噬细胞 （BMM） 为临床的案例研究显示，两种细胞类型之间主要底物的利用率不同。

在本例中，CO2 捕获法用于比较原代颅骨前成细胞的底物氧化与 BBM，后者通常分别用于 体外 成骨细胞或破骨细胞分化。在cMEMα中传代和培养过夜后，它们通常达到汇合的80-90%，并表现出其特征形态。颅内前成骨细胞明显大于BMM（图2）。按照步骤4.1至4.7对每种细胞类型进行葡萄糖，谷氨酰胺和油酸盐的氧化测定。使用等式（1）分析结果。
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Watch this Scientific Journal Video about Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping at JoVE.com

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping

该协议描述了一种易于使用的方法，通过跟踪14CO2的体外生产来检查底物氧化。

使用14CO2捕获在体外评估能量底物氧化

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport&#160;chain (ETC), which generate adenosine triphosphate ...

使用不同的能量底物既是细胞类型特异性的，也是疾病的指示性。测量特定底物的消耗量可以揭示正常的生物学和病理学。该技术不需要专门的设备，并且易于扩展。它也比以前发布的方法更易于使用。了解底物氧化速率的变化将允许开发代谢紊乱的饮食和药物干预。虽然在骨组织中得到了证明，但该技术很容易定制，以研究所有细胞类型的代谢灵活性，从而了解代谢转移的原因和后果。在牺牲幼崽产后三至五天后，将其转移到含有青霉素链霉素双抗生素溶液的冰冷D PBS中。通过去除皮肤和软组织来暴露颅骨。通过在D PBS中从后到前切割周围组织来收集中间区域。用镊子轻轻划伤颅骨的内表面和外表面，以帮助在随后的消化时释放细胞。在D PBS中制备每毫升2和4毫克的二型胶原酶溶液，并用新鲜的0.22微米过滤器过滤每个溶液。首先，用每毫升两毫克胶原酶溶液消化清洁的颅骨15分钟，然后丢弃消化溶液。接下来，将干净的样品在每毫升四毫克胶原酶溶液中消化三次，每次15分钟。然后，拉动消解液并保存。通过70微米细胞过滤器过滤消化溶液，并将滤液在300 RCF离心五分钟。将细胞沉淀重新悬浮在含有10%FBS和青霉素链霉素双抗生素溶液的完全MEMα培养基中。将细胞计数并接种在10厘米的平板中。将细胞在37摄氏度的培养箱中用5%二氧化碳培养三天。然后，在37摄氏度下用0.25胰蛋白酶EDTA解离细胞。消化后，用含有10%FBS和青霉素链霉素双抗生素溶液的完整MEM α培养基在24孔细胞培养板中计数和接种细胞。每个底物的每个细胞类型至少接种四个孔，每个细胞类型至少接种三个额外的孔，用于预测定计数。将细胞在37摄氏度下培养过夜。从八周龄的小鼠身上取出肌肉和结缔组织并收集股骨和胫骨后，用锋利的剪刀切割并丢弃骨骼的两端。将骨髓从一个股骨和一个胫骨冲洗到新鲜的10厘米培养皿中，注射器装有23号针头，其中含有15毫升完整的MEMα培养基，10%FBS青霉素链霉素双抗生素溶液和10%CGM 14 12条件培养基。在培养皿中用5%二氧化碳培养37摄氏度的培养箱中培养细胞。三天后，丢弃培养基并用D PBS冲洗细胞。在37摄氏度下用0.25%胰蛋白酶EDTA解离附着的细胞五分钟。离心后，将细胞沉淀重新悬浮在BMM培养基中。按照前面描述的程序在24孔细胞培养板中计数并接种细胞。在开始测定之前，在37摄氏度下在BMM培养基中培养过夜。用D PBS洗涤额外孔中的细胞两次。用0.25%胰蛋白酶EDTA解离细胞。在D PBS中重新悬浮20微升消化的细胞。用20微升吖啶橙和碘化丙啶染料溶液，用自动细胞计数器确定活细胞的数量并记录数量。用D PBS洗涤测定孔中的细胞两次。向RAM指定的组织培养罩中的每个测定孔中加入500微升热培养基。用parafilm密封板后，将细胞在37摄氏度的RAM指定培养箱中孵育4小时。在孵育过程中，将滤纸切成圆形片，略大于1.5毫升微量离心管盖内的面积，并将纸紧紧地插入盖中。向每个管中加入200微升1摩尔高氯酸，向盖子内的滤纸中加入20微升氢氧化钠。孵育细胞后，将400微升培养基从每个孔转移到制备的试管中并立即关闭盖子。将试管在室温下放入管架中一小时。在孵育过程中，Parallelae为每个管子设置一个闪烁小瓶，并用四毫升闪烁流体填充它。将每张滤纸转移到闪烁小瓶中，并在室温下孵育30分钟。在组织培养罩、水浴、冰箱、培养箱、水槽、地面和任何其他工作区域进行擦拭测试，以发现潜在的RAM污染。将纸擦拭布放入含有闪烁液的闪烁小瓶中。使用闪烁计数器测量闪烁小瓶中的碳14无线电活性并记录读数结果。如有必要，根据辐射安全指南对工作环境进行净化。该图比较了原代颅骨成骨细胞前细胞与BMM的底物氧化。在原代细胞在完整的MEM α培养基中传代和培养过夜后，它们通常达到80%至90%汇合并表现出其特征形态。颅变成骨前细胞明显大于BMM。底物氧化的结果表明，在颅变成骨前体中，每种底物的氧化速率显着高于BMM，可能表明在成骨细胞前通过氧化磷酸化产生更高的能量。插入的第三张纸应略大于1.7毫升的埃宾顶管盖。该方法可以与氧气消耗结合使用，并确定细胞中氧化磷酸化和乳酸产生的总速率。该协议可用于在不同的分化阶段或疾病设置期间确定底物的使用。有了这些知识，我们可以开发代谢紊乱的干预措施。

Research

JoVE Journal

Biology

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

科研

生物学

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Gene Trapping Using Gal4 in Zebrafish

基因捕获斑马鱼中使用的Gal4

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

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

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 ...

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

使用大西洋鲑鱼锌示例证明的补充方法评估鱼类饲料中的矿物供应情况

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

测量重组透气溶性细胞中线粒体底物通量 O-透化细胞

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or ...

Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice

确定肥胖小鼠的基础能量消耗和产热脂肪细胞消耗能量的能力

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...