Name: measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated mouse hepatocytes
Uploaded: 2021-09-09T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Fatty Acid ÃŽÂ²-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes at JoVE.com

这项工作得到了美国国立卫生研究院（National Institutes of Health）向罗伯塔·莱昂纳迪（Roberta Leonardi）授予R35GM119528的支持。

小鼠（C57BL / 6J，雄性，9-11周龄）的所有实验程序均已获得西弗吉尼亚大学机构动物护理和使用委员会（IACUC）的批准。
1. 肝细胞分离
<ol><li>制备<ol>
<li>在肝细胞分离前几天，制备 表1中列出的缓冲液和细胞培养基。设置一个水浴，温度设置为37°C，靠近将要进行手术的地方。</li>
		<li>在肝细胞分离当天，在层流罩下，将35mL缓冲液1转移到无菌的50mL离心管中，将70...

<ol>
	<li>Alves-Bezerra, M., Cohen, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triglyceride+Metabolism+in+the+Liver.">Triglyceride Metabolism in the Liver.</a> Comprehensive Physiology. 8 (1), 1-8 (2017).</li><li>Lopaschuk, G. D., Ussher, J. R., Folmes, C. D., Jaswal, J. S., Stanley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+fatty+acid+metabolism+in+health+and+disease.">Myocardial fatty acid metabolism in health and disease.</a> Physiological Reviews. 90 (1), 207-258 (2010).</li><li>Mannaerts, G. P., van Veldhoven, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+organization+of+peroxisomal+beta-oxidation.">Functions and organization of peroxisomal beta-oxidation.</a> Annals of the New York Academy of Sciences. 804, 99-115 (1996).</li><li>Kerner, J., Hoppel, C. <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+import+into+mitochondria.">Fatty acid import into mitochondria.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1486 (1), 1-17 (2000).</li><li>Baker, 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=Peroxisomal+ABC+transporters:+functions+and+mechanism.">Peroxisomal ABC transporters: functions and mechanism.</a> Biochemical Society Transactions. 43 (5), 959-965 (2015).</li><li>Leonardi, R., Rehg, J. E., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pantothenate+kinase+1+is+required+to+support+the+metabolic+transition+from+the+fed+to+the+fasted+state.">Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state.</a> PloS One. 5 (6), 11107 (2010).</li><li>Shumar, S. A., Kerr, E. W., Fagone, P., Infante, A. M., Leonardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Nudt7+decreases+bile+acid+levels+and+peroxisomal+fatty+acid+oxidation+in+the+liver.">Overexpression of Nudt7 decreases bile acid levels and peroxisomal fatty acid oxidation in the liver.</a> Journal of Lipid Research. 60 (5), 1005-1019 (2019).</li><li>Richert, L., 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=Gene+expression+in+human+hepatocytes+in+suspension+after+isolation+is+similar+to+the+liver+of+origin,+is+not+affected+by+hepatocyte+cold+storage+and+cryopreservation,+but+is+strongly+changed+after+hepatocyte+plating.">Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating.</a> Drug Metabolism and Disposition: The Biological Fate of Chemicals. 34 (5), 870-879 (2006).</li><li>Colbert, R. A., Amatruda, J. M., Young, 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=Changes+in+the+expression+of+hepatocyte+protein+gene-products+associated+with+adaptation+of+cells+to+primary+culture.">Changes in the expression of hepatocyte protein gene-products associated with adaptation of cells to primary culture.</a> Clinical Chemistry. 30 (12), 2053-2058 (1984).</li><li>Spurway, T. D., Sherratt, H. A., Pogson, C. I., Agius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flux+control+coefficient+of+carnitine+palmitoyltransferase+I+on+palmitate+beta-oxidation+in+rat+hepatocyte+cultures.">The flux control coefficient of carnitine palmitoyltransferase I on palmitate beta-oxidation in rat hepatocyte cultures.</a> Biochemical Journal. 323, 119-122 (1997).</li><li>Consitt, L. 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=Peroxisome+proliferator-activated+receptor-gamma+coactivator-1alpha+overexpression+increases+lipid+oxidation+in+myocytes+from+extremely+obese+individuals.">Peroxisome proliferator-activated receptor-gamma coactivator-1alpha overexpression increases lipid oxidation in myocytes from extremely obese individuals.</a> Diabetes. 59 (6), 1407-1415 (2010).</li><li>Lee, S. M., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+hepatocytes+by+a+two-step+collagenase+perfusion+procedure.">Isolation of human hepatocytes by a two-step collagenase perfusion procedure.</a> Journal of Visualized Experiments: JoVE. (79), e50615 (2013).</li><li>Lilly, K., Chung, C., Kerner, J., VanRenterghem, R., Bieber, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+etomoxiryl-CoA+on+different+carnitine+acyltransferases.">Effect of etomoxiryl-CoA on different carnitine acyltransferases.</a> Biochemical Pharmacology. 43 (2), 353-361 (1992).</li><li>Yu, X. X., Drackley, J. K., Odle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+mitochondrial+and+peroxisomal+beta-oxidation+of+palmitate+change+during+postnatal+development+and+food+deprivation+in+liver,+kidney+and+heart+of+pigs.">Rates of mitochondrial and peroxisomal beta-oxidation of palmitate change during postnatal development and food deprivation in liver, kidney and heart of pigs.</a> Journal of Nutrition. 127 (9), 1814-1821 (1997).</li><li>Yu, X. X., Drackley, J. K., Odle, J., Lin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+hepatic+mitochondrial+and+peroxisomal+beta-oxidation+to+increasing+palmitate+concentrations+in+piglets.">Response of hepatic mitochondrial and peroxisomal beta-oxidation to increasing palmitate concentrations in piglets.</a> Biology of the Neonate. 72 (5), 284-292 (1997).</li><li>Veerkamp, J. H., van Moerkerk, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+fatty+acid+oxidation+in+rat+and+human+tissues.+Effect+of+nutritional+state,+clofibrate+treatment+and+postnatal+development+in+the+rat.">Peroxisomal fatty acid oxidation in rat and human tissues. Effect of nutritional state, clofibrate treatment and postnatal development in the rat.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 875 (2), 301-310 (1986).</li><li>Hakvoort, T. 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=Interorgan+coordination+of+the+murine+adaptive+response+to+fasting.">Interorgan coordination of the murine adaptive response to fasting.</a> Journal of Biological Chemistry. 286 (18), 16332-16343 (2011).</li><li>Sokolovic, 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=The+transcriptomic+signature+of+fasting+murine+liver.">The transcriptomic signature of fasting murine liver.</a> BMC Genomics. 9, 528 (2008).</li><li>Kersten, 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=Peroxisome+proliferator-activated+receptor+alpha+mediates+the+adaptive+response+to+fasting.">Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.</a> Journal of Clinical Investigation. 103 (11), 1489-1498 (1999).</li><li>Li, W. C., Ralphs, K. L., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+hepatocytes.">Isolation and culture of adult mouse hepatocytes.</a> Methods in Molecular Biology. 633, 185-196 (2010).</li><li>Ng, I. C., 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=Isolation+of+Primary+Rat+Hepatocytes+with+Multiparameter+Perfusion+Control.">Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control.</a> Journal of Visualized Experiments: JoVE. (170), e62289 (2021).</li><li>Shen, L., Hillebrand, A., Wang, D. Q., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+hepatic+cells.">Isolation and primary culture of rat hepatic cells.</a> Journal of Visualized Experiments: JoVE. (64), e3917 (2012).</li><li>Fulgencio, J. P., Kohl, C., Girard, J., Pegorier, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+metformin+on+fatty+acid+and+glucose+metabolism+in+freshly+isolated+hepatocytes+and+on+specific+gene+expression+in+cultured+hepatocytes.">Effect of metformin on fatty acid and glucose metabolism in freshly isolated hepatocytes and on specific gene expression in cultured hepatocytes.</a> Biochemical Pharmacology. 62 (4), 439-446 (2001).</li><li>Leonardi, R., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pank1+deletion+in+leptin-deficient+mice+reduces+hyperglycaemia+and+hyperinsulinaemia+and+modifies+global+metabolism+without+affecting+insulin+resistance.">Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.</a> Diabetologia. 57 (7), 1466-1475 (2014).</li><li>Bougarne, N., 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=PPARalpha+blocks+glucocorticoid+receptor+alpha-mediated+transactivation+but+cooperates+with+the+activated+glucocorticoid+receptor+alpha+for+transrepression+on+NF-kappaB.">PPARalpha blocks glucocorticoid receptor alpha-mediated transactivation but cooperates with the activated glucocorticoid receptor alpha for transrepression on NF-kappaB.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (18), 7397-7402 (2009).</li><li>Korelova, K., Jirouskova, M., Sarnova, L., Gregor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+3D+collagen+sandwich+culture+of+primary+mouse+hepatocytes+to+study+the+role+of+cytoskeleton+in+bile+canalicular+formation+in+vitro.">Isolation and 3D collagen sandwich culture of primary mouse hepatocytes to study the role of cytoskeleton in bile canalicular formation in vitro.</a> Journal of Visualized Experiments: JoVE. (154), e60507 (2019).</li></ol>

在肝脏灌注期间，避免引入气泡至关重要，因为它们会阻塞肝脏中的微毛细血管，防止或限制缓冲液循环并总体上降低肝细胞产量和活力20，21。预防措施，例如在IVC插管之前仔细检查填充缓冲液的入口管路，并避免将入口管从含有缓冲液1的管中抬起以切换到缓冲液2，如本文所述，可以成功减少失败灌注的数量（活力<70%）。在灌注系统中使用气泡捕集?...

脂肪酸β氧化是脂质代谢的必要过程，提供分解代谢途径来平衡脂肪酸合成和饮食中的摄入量。该过程为多个器官产生能量，包括心肌，肾皮层和禁食肝脏，并利用从饮食中获得的脂肪酸，脂肪组织脂肪分解和内部甘油三酯储存1，2。
脂肪酸通过β氧化途径的氧化导致脂肪酰基链一次由两个碳连续缩短，以乙酰辅酶A的形式释放，并且该过程同时发生在线粒体和过氧化物酶体中。虽然大多数脂肪酸只经历β氧化，但有些脂肪酸在进入该途径之前在不同的碳中被氧化。例如，3-甲基取代的脂肪酸，如植烷酸，在进入β氧化途径之前，通过过氧化物酶体中的α氧化除去一个碳。类似地，一些脂肪酸首先通过氧化内质网中的末端甲基（ω-氧化）转化为二羧基脂肪酸，然后通过β氧化3在过氧化物酶体中优先氧化。
无论特定的细胞器如何，脂肪酸必须首先转化为辅酶A（CoA）硫酯或酰基辅酶A，才能通过β氧化途径被氧化。线粒体基质中长链酰基辅酶A的β氧化需要肉碱穿梭进行其易位，其中肉碱棕榈酰转移酶1（CPT1）催化酰基辅酶A向酰基肉碱的转化，并且是该过程中的限速酶4。一旦转移到线粒体基质，酰基辅酶A被重新形成并作为线粒体β氧化机制的底物。在禁食状态下，肝线粒体中通过β氧化产生的乙酰辅酶A主要引导至生酮作用。过氧化物酶体是超长链，支链和二羧酸脂肪酸β氧化的主要位点。过氧化物酶体不需要肉碱穿梭来导入脂肪酸底物，而是通过ATP结合盒（ABC）转运蛋白ABCD1-3的活性导入相应的酰基辅酶A5。在过氧化物酶体内，酰基辅酶A随后被一组专用的酶氧化，这与线粒体脂肪酸β氧化机制不同。线粒体和过氧化物酶体也需要供应NAD + 和游离辅酶A来氧化脂肪酰基链。肝脏中的CoA水平已被证明随着空腹而增加，支持在这种状态下发生的脂肪酸氧化速率的增加6。此外，过氧化物酶体中CoA降解的增加导致过氧化物酶体脂肪酸氧化7的选择性降低。因此，细胞内脂肪酸氧化的过程受到参与脂肪酸活化，运输和氧化的酶的表达水平和活性的调节，以及整个多个亚细胞区室的辅助因子和其他代谢物的浓度。
使用组织匀浆来测量脂肪酸氧化的程序破坏了调节和支持该过程的细胞结构，导致数据收集不能准确反映体内代谢。虽然使用接种的原代肝细胞的技术保留了该系统，但长时间培养分离的细胞会导致细胞中仍然存在的体内基因表达谱的丧失，当它们仍然生活在动物体内时8，9。以下方案描述了一种分离原代肝细胞的方法，并使用[1-14C]棕榈酸在分离后立即和悬浮液测定其脂肪酸β氧化的能力。该测定基于测量与酸溶性代谢物（ASM）或产物（如乙酰辅酶A）相关的放射性，由[1-14 C]棕榈酸10，11的β氧化产生。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(R)-(+)-Etomoxir sodium salt</td>
			<td>Tocris Bioscience</td>
			<td>4539/10</td>
			<td></td>
		</tr>
		<tr>
			<td>[1-14C]-Palmitic acid, 50&#8211;60 mCi/mmol, 0.5 mCi/mL</td>
			<td>American Radiolabeled Chemicals</td>
			<td>ARC 0172A</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES, sterile</td>
			<td>Corning</td>
			<td>25060CI</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L disposable capillaries/pistons for positive displacement pipette</td>
			<td>Mettler Toledo</td>
			<td>17008604</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L, 200 &#181;L, and 10 &#181;L pipettes and tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL, 10 mL, and 25 mL serological pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>CellTreat</td>
			<td>229421</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Perchloric acid</td>
			<td>Fisher Scientific</td>
			<td>A2296-1LB</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, fatty acid-free</td>
			<td>Fisher Scientific</td>
			<td>BP9704100</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 dihydrate</td>
			<td>MilliporeSigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>MilliporeSigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Gold Biotechnology</td>
			<td>E-217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco</td>
			<td>111000200CSPP</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System, 0.22 &#956;m PES Filter, 500 mL, Sterile</td>
			<td>CellTreat</td>
			<td>229707</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulphate</td>
			<td>Gold Biotechnology</td>
			<td>G-400-25</td>
			<td></td>
		</tr>
		<tr>
			<td>HDPE, 6.5 mL scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>03-342-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needles 22 G, 1.5 in</td>
			<td>BD Biosciences</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VetOne</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MilliporeSigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TM Research Grade</td>
			<td>MilliporeSigma</td>
			<td>5401119001</td>
			<td>Defined blend of purified collagenase I and II with a medium concentration of thermolysin</td>
		</tr>
		<tr>
			<td>M199 medium</td>
			<td>MilliporeSigma</td>
			<td>M5017</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 heptahydrate</td>
			<td>MilliporeSigma</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>accuSpin Micro 17</td>
		</tr>
		<tr>
			<td>Microdissecting Scissors</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>RS-5980</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chem-Impex International</td>
			<td>30070</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Acros Organics</td>
			<td>424270010</td>
			<td></td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>MilliporeSigma</td>
			<td>P0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin (100x)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive displacement pipette MR-10, 10 &#181;L</td>
			<td>Mettler Toledo</td>
			<td>17008575</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge with inserts for 50 mL conical tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>5810 R</td>
		</tr>
		<tr>
			<td>Round-bottom, 14 mL, polypropylene culture test tubes</td>
			<td>Fisher Scientific</td>
			<td>14-956-9A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>TriCarb 4810 TR</td>
		</tr>
		<tr>
			<td>ScintiVerse BD cocktail</td>
			<td>Fisher Scientific</td>
			<td>SX18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking water bath, 30 L capacity</td>
			<td>New Brunswick Scientific</td>
			<td></td>
			<td>&#160;Model G76</td>
		</tr>
		<tr>
			<td>Sterile cell strainers, 100 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Thumb Dressing Forceps</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>RS-8120</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>25900CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable-flow peristaltic pump</td>
			<td>Fisher Scientific</td>
			<td>138762</td>
			<td></td>
		</tr>
		<tr>
			<td>Water baths, 2&#8211;2.5 L capacity</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated mouse hepatocytes

脂肪酸β氧化是满足肝脏能量需求的关键代谢途径，并为其他过程（如生酮和糖异生）提供底物和辅助因子，这对于维持全身葡萄糖稳态和支持禁食状态下的肝外器官功能至关重要。脂肪酸β氧化发生在线粒体和过氧化物酶体内，并通过多种机制进行调节，包括脂肪酸的摄取和活化，酶表达水平以及辅酶A和NAD +等辅助因子的可用性。在测量肝脏匀浆中脂肪酸β氧化的测定中，细胞裂解和辅助因子的超生理水平的常见添加掩盖了这些调节机制的影响。此外，匀浆中细胞器的完整性难以控制，并且在制剂之间可能会有很大差异。完整原代肝细胞中脂肪酸β氧化的测量克服了上述缺陷。该协议描述了一种测量与 14个C标记棕榈酸一起孵育的新鲜分离的原代小鼠肝细胞悬浮液中脂肪酸β氧化的方法。通过避免数小时至数天的培养，该方法具有更好地保存原始肝脏的蛋白质表达水平和代谢途径活性的优点，包括与喂养小鼠相比，在从禁食小鼠分离的肝细胞中观察到的脂肪酸β氧化的活化。

这里描述的肝脏灌注通常产生3000-4000万个细胞/肝脏，平均活力为80%，通过台盼蓝排除估计（图2）。用于制备灌注缓冲液1和2的克雷布斯 - 亨赛莱特缓冲液（KHB）中葡萄糖的典型浓度为11 mM。当测量从禁食小鼠分离的肝细胞中的脂肪酸β氧化时，可以降低KHB中葡萄糖的浓度以更好地代表禁食状态。如图 2所示，将葡萄糖浓度降低到5.6mM对肝细胞的产量或活?...

Watch this Scientific Journal Video about Measurement of Fatty Acid ÃŽÂ²-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes at JoVE.com

Measurement of Fatty Acid ÃƒÅ½Ã‚Â²-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

脂肪酸β氧化是负责在许多不同细胞类型（包括肝细胞）中产生能量的重要代谢途径。在这里，我们描述了一种使用 14个C标记的棕榈酸测量新鲜分离的原代肝细胞中脂肪酸β氧化的方法。

测量新鲜分离的小鼠肝细胞悬浮液中的脂肪酸β氧化

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

该方案的开发旨在为测量原代小鼠肝细胞中的总线粒体和过氧化物酶体脂肪酸β-氧化提供可靠的方法。使用完整细胞可以维持细胞器的完整性和调节机制。此外，检测新鲜分离的肝细胞可最大限度地减少基因表达相对于原产地肝脏的变化。手术可能具有挑战性。你必须自信、勤奋和专注。一旦插管成功，尝试放松，但要注意并监测灌注的质量。要开始该过程，请用70%乙醇自由喷洒麻醉小鼠的腹部和胸部。然后使用镊子拉起靠近腹部底部的皮肤和腹壁，并在中线的两侧横向切开，直到横膈膜以暴露器官。暴露下腔静脉或IVC，通过将肠道移动到右侧并轻轻地向上翻转肝脏叶。在鼠标背面下方插入一个小圆柱形物体，例如针帽，以稍微倾斜IVC并促进插管。以最低速度启动带有缓冲液一的泵后，将针插入IVC。然后切开门静脉以减轻压力并允许血液和灌注缓冲液的引流。在将流速增加到每分钟7毫升之前。用温缓冲液1灌注肝脏，并确保插入含有缓冲液1的管中的管线保持连续浸没状态，以避免引入气泡。在进行灌注时，加入130微升胶原酶溶液以缓冲液2，并通过用血清移液管移液混合。当含有缓冲液一的试管中的体积减少到约5mL时，通过在试管侧面移液，缓慢加入5 mL缓冲液2至缓冲液1。重复添加两次，然后将剩余的缓冲液二加入管中。当约5至10mL缓冲液2留在管中时停止灌注。然后切除肝脏并将其转移到含有20mL冰冷缓冲液的100mL培养皿中。在层流罩下，使用手术剪刀和镊子轻轻地将肝组织分开。接下来，向肝细胞悬浮液中加入约20mL冰冷的M199缓冲液，并使用注射器的柱塞通过100微米细胞过滤器过滤，以轻轻促进从较大的肝脏碎片中释放额外的肝细胞。用M199缓冲液洗涤100mL培养皿和细胞过滤器以收集细胞悬浮液，直到收集管充满。然后在4摄氏度下以50×g离心悬浮液两分钟。吸出上清液，然后通过旋转将肝细胞沉淀重悬于30mL冷M199中。重复洗涤一次。在制备用于多种反应的底物混合物之前，解冻棕榈酸和牛血清白蛋白或BSA溶液。在微量离心管中每次反应等分13.5微升BSA溶液。将管加热至41摄氏度后，每次反应加入一微升200mM棕榈酸溶液。在41摄氏度孵育之前和期间，大力涡旋管20至30分钟，以促进可溶性棕榈酸 - BSA复合物的形成。在此期间，等分试样133微升1M高氯酸，其将用于在单独的1.5mL微量离心管中停止反应。在开始反应之前，等分试样并将每次反应的485.5微升M199缓冲液孵育到37摄氏度的管中以稀释制备的放射性BSA-棕榈酸复合物。接下来，在单独的14 mL圆底管中分配750微升M199（含或不带抑制剂）作为样品。在肝细胞洗涤步骤中，在开始反应前10至15分钟，将管子转移到设置为37摄氏度的振荡水浴中，每分钟旋转180至200。如果肝细胞的活力至少为75%，则通过将每次反应0.8微升碳-14棕榈酸转移到含有澄清的BSA-棕榈酸溶液的微量离心管中来完成底物混合物的制备。在将管子返回到41摄氏度的水浴之前进行涡旋。在最后一次肝细胞重悬后，立即使用一mL移液将750微升肝细胞悬浮液转移到振荡水浴中的每个14mL圆底管中，并在转移到培养箱之前以低速短暂涡旋管，将每次添加间隔30秒。将单独的肝细胞等分试样转移到1.5mL微量离心管中，以3000×g旋转5分钟。在将沉淀以零下80摄氏度储存之前除去上清液，以测量样品中蛋白质的总量以使结果正常化。当肝细胞在37摄氏度下预孵育时，将放射性BSA-棕榈酸复合物加入温暖的培养基中以保持在37摄氏度，直到准备好开始反应。要开始反应，从水浴中取出肝细胞，并向肝细胞中加入500微升底物混合物。以低速涡旋细胞五秒钟，然后返回水浴孵育15分钟。启动一组反应并立即停止以确定背景放射性。转移并留出200至250微升剩余底物混合物的重复等分试样在6-mL闪烁小瓶中进行计数。为了停止反应，从水浴中取出肝细胞以以中等速度涡旋重悬，然后将400微升肝细胞悬浮液转移到含有高氯酸的微量离心管中。在对所有样品重复序列之前，立即盖上并涡旋试管，错开30秒。在13， 000×g下旋转1.5mL微量离心管10分钟后，将300微升上清液转移到6mL闪烁小瓶中，并加入4mL闪烁液以计数样品中的放射性，并在闪烁计数器中将底物混合物等分试样计数。在研究中，肝脏灌注每个肝脏产生3000万至4000万个细胞，平均活力为80%，还观察到降低空腹组的葡萄糖浓度对肝细胞的产量或活力没有负面影响。从喂养和禁食的雄性小鼠中分离的肝细胞悬浮液在存在和不存在etomoxir（CPT1和线粒体脂肪酸氧化的有效抑制剂）的情况下测定脂肪酸β-氧化能力。与背景放射性相关的每分钟计数或CPM随棕榈酸批次而变化。然而，CPM仍然显着低于与底物混合物一起孵育的样品。从禁食小鼠分离的肝细胞显示出线粒体和过氧化物酶体脂肪酸β-氧化速率的显着增加。进一步研究了数据以确定从喂养和禁食小鼠分离的肝细胞中棕榈酸被氧化的速率。防止气泡进入线。在灌注期间保持针头稳定。处理肝细胞时要温和。分配时，请将其悬架。从该程序中分离的肝细胞可以用作悬浮液来测定其他代谢途径，例如脂肪酸合成，或者它们可以接种用于细胞培养实验。

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Education

JoVE Journal

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Cell Biology

Biochemistry

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Unit 1

Introduction to Metabolism

SCIENTISTS IN ACTION

Identification of Fatty Acids in Bacillus cereus

Identification of Fatty Acids in Bacillus cereus

脂肪酸鉴定<em&gt;蜡状芽孢杆菌</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

科研

生物化学

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

镍 - 席夫碱的水解复杂环境条件适宜酸不稳定保护基团的保留

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...