Name: isolation and differentiation of primary white and brown preadipocytes from newborn mice
Uploaded: 2021-01-25T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice at JoVE.com

作者感谢西班牙马德里国家生物技术中心的克里斯蒂娜·戈迪奥、斯克里普斯研究所的玛丽·甘特纳、巴尔的摩约翰霍普金斯大学的阿纳斯塔西娅·克拉利在卡恩等人的最初工作基础上帮助优化了这一协议。这项工作由NIH向E.S.提供DK114785和DK121196资助。

该协议遵循了斯克里普斯研究所和威斯康星大学麦迪逊医学院和公共卫生学院的所有 IACUC 指南。
1. 脂肪库的收集和消化（第1天）
<ol><li>为每只小狗准备两个 1.5 mL 管：一个用于棕色脂肪组织 （BAT），一个用于白色脂肪组织 （WAT）。添加 250 μL 磷酸盐缓冲盐水 （PBS） + 200 μL 的 2 倍隔离缓冲（123 mM NaCl， 5 mM KCl， 1.3 mM CaCl2， 5 mM 葡萄糖， 100 mM 4-（2-羟基苯甲基） - 1...

<ol>
	<li>Rosen, E. D., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+we+talk+about+when+we+talk+about+fat.">What we talk about when we talk about fat.</a> Cell. 156 (1-2), 20-44 (2014).</li><li>McGarry, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Banting+lecture+2001:+Dysregulation+of+fatty+acid+metabolism+in+the+etiology+of+type+2+diabetes.">Banting lecture 2001: Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes.</a> Diabetes. 51 (1), 7-18 (2002).</li><li>Kusminski, C. M., Bickel, P. E., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+adipose+tissue+in+the+treatment+of+obesity-associated+diabetes.">Targeting adipose tissue in the treatment of obesity-associated diabetes.</a> Nature Reviews Drug Discovery. 15 (9), 639-660 (2016).</li><li>Waki, H., 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+small+molecule+harmine+is+an+antidiabetic+cell-type-specific+regulator+of+PPARgamma+expression.">The small molecule harmine is an antidiabetic cell-type-specific regulator of PPARgamma expression.</a> Cell Metabolism. 5 (5), 357-370 (2007).</li><li>Dominguez, 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=Integrated+phenotypic+and+activity-based+profiling+links+Ces3+to+obesity+and+diabetes.">Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes.</a> Nature Chemical Biology. 10 (2), 113-121 (2014).</li><li>Galmozzi, 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=ThermoMouse:+an+in+vivo+model+to+identify+modulators+of+UCP1+expression+in+brown+adipose+tissue.">ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue.</a> Cell Reports. 9 (5), 1584-1593 (2014).</li><li>Ye, 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=TRPV4+is+a+regulator+of+adipose+oxidative+metabolism,+inflammation,+and+energy+homeostasis.">TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis.</a> Cell. 151 (1), 96-110 (2012).</li><li>Lynes, M. D., Tseng, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+adipose+tissue+heterogeneity.">Deciphering adipose tissue heterogeneity.</a> Annals of the New York Academy of Sciences. 1411 (1), 5-20 (2018).</li><li>Schoettl, T., Fischer, I. P., Ussar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+adipose+tissue+in+development+and+metabolic+function.">Heterogeneity of adipose tissue in development and metabolic function.</a> Journal of Experimental Biology. 221 (PT Suppl 1), jeb162958 (2018).</li><li>Sponton, C. H., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+roles+of+beige+fat+in+energy+homeostasis+beyond+UCP1.">Multifaceted roles of beige fat in energy homeostasis beyond UCP1.</a> Endocrinology. 159 (7), 2545-2553 (2018).</li><li>Gao, W., Kong, X., Yang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+primary+culture,+and+differentiation+of+preadipocytes+from+mouse+brown+adipose+tissue.">Isolation, primary culture, and differentiation of preadipocytes from mouse brown adipose tissue.</a> Methods in Molecular Biology. 1566, 3-8 (2017).</li><li>Yu, H., Emont, M., Jun, H., Wu, J. <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+differentiation+of+murine+primary+brown/beige+preadipocytes.">Isolation and differentiation of murine primary brown/beige preadipocytes.</a> Methods in Molecular Biology. (1773), 273-282 (2018).</li><li>Church, C. D., Berry, R., Rodeheffer, M. S. <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+study+of+adipocyte+precursors.">Isolation and study of adipocyte precursors.</a> Methods in Enzymology. (537), 31-46 (2014).</li><li>Hausman, D. B., Park, H. J., Hausman, G. J. <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+preadipocytes+from+rodent+white+adipose+tissue.">Isolation and culture of preadipocytes from rodent white adipose tissue.</a> Methods in Molecular Biology. (456), 201-219 (2008).</li><li>Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., Frisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122 (1), 133-143 (2005).</li><li>Spalding, K. 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=Dynamics+of+fat+cell+turnover+in+humans.">Dynamics of fat cell turnover in humans.</a> Nature. 453 (7196), 783-787 (2008).</li><li>Wang, Q. A., Tao, C., Gupta, R. K., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+adipogenesis+during+white+adipose+tissue+development,+expansion+and+regeneration.">Tracking adipogenesis during white adipose tissue development, expansion and regeneration.</a> Nature Medicine. 19 (10), 1338-1344 (2013).</li><li>Kahn, C. R. <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+brown+fat+preadipocytes.">Isolation and primary culture of brown fat preadipocytes.</a> Sprotocols. , (2014).</li><li>Mills, E. 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=Accumulation+of+succinate+controls+activation+of+adipose+tissue+thermogenesis.">Accumulation of succinate controls activation of adipose tissue thermogenesis.</a> Nature. 560 (7716), 102-106 (2018).</li><li>Kajimura, S., Spiegelman, B. M., Seale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+and+beige+fat:+physiological+roles+beyond+heat+generation.">Brown and beige fat: physiological roles beyond heat generation.</a> Cell Metabolism. 22 (4), 546-559 (2015).</li><li>Pydi, 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=Adipocyte+&#946;-arrestin-2+is+essential+for+maintaining+whole+body+glucose+and+energy+homeostasis.">Adipocyte &#946;-arrestin-2 is essential for maintaining whole body glucose and energy homeostasis.</a> Nature Communications. 10 (1), 2936 (2019).</li><li>Sun, 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=snRNA-seq+reveals+a+subpopulation+of+adipocytes+that+regulates+thermogenesis.">snRNA-seq reveals a subpopulation of adipocytes that regulates thermogenesis.</a> Nature. 587 (7832), 98-102 (2020).</li><li>Galmozzi, 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=PGRMC2+is+an+intracellular+haem+chaperone+critical+for+adipocyte+function.">PGRMC2 is an intracellular haem chaperone critical for adipocyte function.</a> Nature. 576 (7785), 138-142 (2019).</li><li>Brown, E. 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=Estrogen-related+receptors+mediate+the+adaptive+response+of+brown+adipose+tissue+to+adrenergic+stimulation.">Estrogen-related receptors mediate the adaptive response of brown adipose tissue to adrenergic stimulation.</a> iScience. 2, 221-237 (2018).</li><li>Shinoda, K., 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=Genetic+and+functional+characterization+of+clonally+derived+adult+human+brown+adipocytes.">Genetic and functional characterization of clonally derived adult human brown adipocytes.</a> Nature Medicine. 21 (4), 389-394 (2015).</li><li>Wang, Q. A., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AdipoChaser+mouse:+A+model+tracking+adipogenesis+in+vivo.">The AdipoChaser mouse: A model tracking adipogenesis in vivo.</a> Adipocyte. 3 (2), 146-150 (2014).</li></ol>

脂肪组织对全身胰岛素敏感性和葡萄糖平衡20至关重要。肥胖相关脂肪细胞功能障碍与2型糖尿病的发病密切相关。因此，对脂肪组织的基本生物学和生理学的更深入的了解，可能有助于设计代谢紊乱的新疗法。作为对从脂肪库分离出来的成熟脂肪细胞的直接功能和转录分析的补充，培养的原发性脂肪细胞已被证明可以概括脂肪组织病?...

肥胖症源于能量摄入和能量消耗之间的长期不平衡。随着肥胖的发展，白色脂肪细胞在细胞大小上经历了巨大的扩张，导致微环境缺氧，细胞死亡，炎症和胰岛素抵抗1。功能失调，肥大的脂肪细胞不能正确地储存多余的脂质，而积聚在其他组织中，它们抑制胰岛素作用2，3。改善脂肪细胞功能和恢复组织间正常脂质分区的制剂，预计对治疗以胰岛素抵抗（如2型糖尿病）为特征的肥胖相关疾病是有益的。使用不朽细胞系（如 3T3-L1、F442A 和 10T 1/2）在脂肪细胞中的表型屏幕已被证明有助于识别调节脂肪生成的遗传因素，并分离具有抗糖尿病特性的亲脂肪分子 4、5、6、7。然而，这些细胞系并不能完全反映脂肪库中细胞类型的异质性，包括具有独特特征的白色、棕色、米色和其他脂肪细胞亚型，所有这些都有助于系统性平衡8、9、10。此外，培养的细胞系通常显示对外部刺激的反应减弱。
相比之下，初级脂肪细胞培养更准确地概括了体内脂肪生成的复杂性，而原发性脂肪细胞则显示出强大的功能反应。主要的预生物细胞通常从成年小鼠11、12、13、14的脂肪库的频闪血管部分分离出来。然而，由于成年动物的脂肪库主要由完全成熟的脂肪细胞组成，其周转率非常缓慢，15、16、17，这种方法产生的预生细胞数量有限，增殖率低。因此，从新生小鼠身上分离出预生细胞比获得大量可在体外分化的快速生长细胞更可取。在这里，一个协议已经描述，灵感来自与卡恩等人的初级棕色脂肪细胞的初始工作，有效地隔离白色和棕色的预生细胞，可以在体外扩展和区分成功能齐全的初级脂肪细胞（图1A）。与成年小鼠相比，将原细胞与新生儿隔离开来的优点是脂肪库正在迅速生长，因此是积极增殖17预生细胞的丰富来源。使用此协议分离的细胞具有很高的扩散能力，能够快速扩展培养物。此外，新生幼崽的预生细胞比成年祖先具有更高的分化潜力，这降低了分化程度的变异性，从而增加了可重复性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>AdipoRed (Nile Red)</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>BenchMark Fetal Bovine Serum</td>
			<td>Gemini Bioproducts LLC</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1&#160;&#160;</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>Sigma-Aldrich</td>
			<td>6442</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D5030</td>
			<td>For Bioenergetics studies</td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, Glutamax</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty Acid-Free BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Norepinephrine</td>
			<td>Cayman Chemical</td>
			<td>16673</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>557368</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent Technologies</td>
			<td>102416-100</td>
			<td>For Bioenergetics studies</td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>ROBOZ Surgical Instrument Co</td>
			<td>RS-5158</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>ROBOZ Surgical Instrument Co</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Eppendorf</td>
			<td>T1317</td>
			<td></td>
		</tr>
		<tr>
			<td>triiodothyronine (T3)</td>
			<td>Sigma-Aldrich</td>
			<td>642511</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and differentiation of primary white and brown preadipocytes from newborn mice

对脂肪细胞分化和功能背后的机制的理解大大受益于不朽的白色前体细胞系的使用。然而，这些培养的细胞系有局限性。它们不能完全捕获异质脂肪细胞种群的不同功能谱，这些细胞群现在已知存在于白色脂肪库中。为了提供一个更生理相关的模型来研究白脂肪组织的复杂性，已经开发和优化了一个协议，使原发性白细胞和棕色脂肪细胞祖先与新生小鼠同时隔离，它们在培养上迅速扩张，并在体外分化成成熟、功能齐全的脂肪细胞。将原细胞与新生儿而不是成年小鼠隔离开来的主要优点是脂肪库正在积极发育，因此是繁殖前细胞的丰富来源。使用此协议分离的主要预生细胞在达到汇合时迅速分化，并在 4-5 天内完全成熟，这是一个时间窗口，可准确反映新生小鼠发育的脂肪垫的外观。使用这种策略准备的主要培养物可以扩展和研究具有很高的可重复性，使其适合遗传和表型屏幕，并能够研究基因小鼠模型的细胞自主脂肪表型。此协议提供了一种简单、快速和廉价的方法，以研究体外脂肪组织的复杂性。

协议第1节将产生在标准光显微镜下可见的细胞的异质悬浮。用细胞过滤器过滤消化的组织（第2节）将去除未消化的组织。然而，一些细胞碎片，血细胞和成熟的脂肪细胞将通过（图1C）。电镀后轻轻洗涤1小时将去除非相关细胞，因为预编细胞会迅速附着在井底（图1C）。在第3节中，脂肪细胞前体被扩展，以获得实验计划所需?...

Watch this Scientific Journal Video about Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice at JoVE.com

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

本报告描述了一个同时将原生棕色和白色预生细胞与新生小鼠隔离的协议。孤立的细胞可以在培养中生长，并诱导分化成完全成熟的白色和棕色脂肪细胞。该方法使培养中原脂肪细胞的遗传、分子和功能特征化。

初白和棕色预生细胞与新生小鼠的隔离和分化

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

该协议提供了一个简单的策略，以产生白色和棕色脂肪细胞的细胞培养模型，忠实地回顾体外，我们在体内的脂肪组织的生理学。该协议允许同时分离来自新生小鼠的白色和棕色预生细胞，以及我们使用这种方法分离的细胞，评估高扩散能力和分化潜力。使用这种方法分离的细胞比其他培养模型更能反映脂肪组织的复杂性。它可以用来更准确地研究肥胖和糖尿病中的脂肪细胞功能。我们主要关注于了解肥胖症中的脂肪组织功能障碍。然而，用这种方法准备的细胞也可以用来研究发育生物学中细胞的基本问题。成功实验的关键是确定如何确定白色脂肪库为这些库，这些仓库虽小，但非常丰富且繁殖的预生细胞在幼崽中很难区分。视觉演示说明此协议是多么简单，并允许提供查找和解剖白色和棕色脂肪库的提示。看到仓库隔离是非常有帮助的。收集皮下白色脂肪组织，切开安乐死的新生幼崽腹部周围的皮肤，轻轻拉下腿部的皮肤。在不收集任何皮肤组织的情况下，小心地收获脂肪库，脂肪库将显示为附着在皮肤内部或四头肌顶部的透明或白色、细拉长组织。冲洗 PBS 中收获的仓库，并将脂肪组织放置在 1.5 毫升管中，其中包含 250 微升 PBS 和 200 微升 2X 隔离缓冲器。要收集闭双面棕色脂肪组织，从肩部刀片上拉起皮肤，抬起位于肩部刀片之间的深红色棕色脂肪组织。小心地在脂肪周围切开，将其从身体分离出来，并检查组织是否一致性和颜色。冲洗后，将组织放在第二个1.5毫升管中，其中含有250微升PBS和200微升的2X隔离缓冲器。收集完所有仓库后，使用剪刀将每个仓库直接在管内轻轻切碎四到六次，并在每个管子的 2 倍隔离缓冲中加入 50 微升 10 倍拼贴酶。然后快速倒置管子，在37摄氏度和每分钟1400转的温度控制搅拌机中混合并放置组织30分钟。在消化结束时，将管子放回冰上，通过100微米细胞过滤器过滤消化组织，进入新的50毫升管。为了最大限度地提高细胞产量，用一毫升的隔离介质冲洗每个管子，并通过过滤器过滤洗涤。在新的隔离介质中，将棕色和白色脂肪组织溶液稀释到每口油井两毫升的组织悬浮液中。然后板两毫升的白色脂肪组织悬浮到每四到六口井和两毫升的棕色脂肪组织悬浮到每两到三口井的六井板通过一个100微米过滤器每井。当所有细胞都镀上后，将板放在细胞培养孵化器中60至90分钟。在孵化结束时，用两毫升无血清介质清洗每口油井，轻轻搅拌板，将任何血细胞从井底分离。洗三次后，在井中加入两毫升新鲜隔离介质，并将板返回细胞培养孵化器。当细胞达到80%至90%的结合度时，在37摄氏度的蒸馏水中涂上无菌0.1%明胶的新目标板，10分钟。当板正在孵化时，用PBS清洗细胞，然后在细胞培养孵化器中用肌素治疗三分钟。当细胞分离时，移液器将细胞悬架多次，以最大限度地提高细胞的恢复，并将细胞转移到新的管子上。收集完所有细胞后，用一毫升的隔离介质清洗每口井，然后用细胞悬架拉洗。通过离心收集细胞，并将颗粒重新用三到五毫升的隔离介质进行计数。将细胞稀释到适当的浓度，用于在新鲜隔离介质中电镀，用 PBS 清洗明胶涂层板，以去除任何多余的明胶。然后将细胞播种到明胶涂层板上，并将细胞返回细胞培养孵化器。当细胞达到共存时，用分化介质替换隔离介质。如果区分棕色脂肪组织前体细胞，也添加一个纳米材料三聚二甲酸酯。48 小时后，用每个培养金适当的维护介质刷新介质。此时，细胞内小液滴的积累应在明亮的场显微镜下可见。即使在过滤后，一些细胞碎片和血细胞仍然留在细胞悬架内。电镀一小时后轻轻洗涤会去除与此无关的细胞，因为预编细胞会迅速附着在井底。虽然从新生幼崽身上分离出的白色和棕色预生细胞具有很高的扩散能力，但每个库的白色预生细胞的产量通常是棕色预生细胞的两倍。因此，如果需要同步培养，则必须相应地计算白色预组织体的启动密度。在分化结束时，细胞将显得充满脂质液滴，并分别表达白色和棕色脂肪细胞的经典标记。在基底条件下初级白细胞和棕色脂肪细胞的线粒体应激测试中，以及针对线粒体功能已知刺激器的对比分析中，可以观察到每个细胞类型的这种特定生物能学能力。请记住，我们正在隔离活细胞，我们希望培养它们几天。因此，请务必在隔离期间保持无菌状态，以避免污染，这可能会损害后续的文化。这种方法产生完全成熟的脂肪细胞，可用于不同的应用，包括功能检测，基因或化学屏幕，或卵母分析，以研究细胞自主机制，影响脂肪细胞功能。

Research

JoVE Journal

Biology

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

从P0新生大鼠海马神经元的原代培养

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

科研

生物学

Isolation of Immune Cells from Primary Tumors

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Unveiling Inflammatory Arthritis&#58; Exploring Functions of Synovial Cells

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue (Video) | JoVE 

小鼠关节炎组织中原代滑膜巨噬细胞和成纤维细胞的分离和培养

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...