Name: isolating brown adipocytes from murine interscapular brown adipose tissue for gene and protein expression analysis
Uploaded: 2021-03-12T14:30:00
Description: Watch this Scientific Journal Video about Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis at JoVE.com

Z. Lin得到了美国国立卫生研究院HL138454-01和共济会医学研究所基金的支持。

所有小鼠均在无病原体条件下饲养，所有程序均由共济会医学研究机构动物护理和使用委员会（IACUC）批准。UCP1：：Cre9 和Rosa 26tdTomato 小鼠系13 之前已报道。将所有小鼠保持在室温下，进行12小时的光照/黑暗循环。
1. 制备溶液和棕色脂肪组织（BAT）
<ol><li>在 15 mL 离心管中制备消解溶液和分离溶液。</li>
	<li>10 mL BAT 消化液：在 10 mL...

<ol>
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本研究开发了一种分离BAs用于基因和蛋白质表达分析的新方法。
在已发布的BAs分离方案中，使用3%BSA溶液来富集BAs12。然而，通过该已发表的方案获得的富集BA不能直接用于蛋白质表达分析。这是因为BAs溶液中存在的浓缩BSA会干扰后续蛋白质提取。当富集在3%BSA溶液中的BA用Trizol试剂处理时，会形成粘性蛋白质聚集体，其中大部分不溶于GTPC蛋白质提取过程。此...

小鼠和人类都有两种类型的脂肪组织：白色脂肪组织（WAT）和棕色脂肪组织（BAT）1。WAT以甘油三酯的形式将能量储存在白色脂肪细胞中，BAT的棕色脂肪细胞（BAs）以热量的形式耗散化学能2。根据其发育起源，BA进一步分为胚胎发育过程中形成的经典BA（cBA）和白色脂肪细胞衍生的BA（米色/白细胞，在应激条件下从白色脂肪细胞转化而来）3。BA是多房的，表达产热蛋白解偶联蛋白1（UCP1）4。肩胛间蝙蝠（iBAT）储存库是小型哺乳动物5的主要cBAs储存库之一，而米色细胞分散在WAT6内。 
由于其耗散能量的性质，BAs作为减少肥胖的治疗靶点而受到广泛关注7。为了利用BA治疗肥胖症，必须了解控制BA功能，存活和募集的分子机制。脂肪组织包括BAT和WAT是异质的。除脂肪细胞外，脂肪组织还含有许多其他细胞类型，例如内皮细胞，间充质干细胞和巨噬细胞8。尽管存在特异性消耗小鼠BA候选基因的遗传工具，例如UCP1：：Cre系9，但从BAT或WAT纯化BA的技术有限，因此难以在单细胞类型水平上研究BA。此外，如果没有获得纯BA，BA和非BA之间的关系将无法明确界定。例如，血小板衍生生长因子受体α（PDGFRα）已被用作未分化间充质细胞的标志物，并在BAT的内皮和间质细胞中表达。在冷应激的BAT中，PDGFRα阳性祖细胞产生新的BAs10。PDGFRα被其配体PDGF激活，PDGF是一种调节间充质细胞生长和增殖的生长因子11;然而，尚不清楚BAs是否通过分泌PDGF影响PDGFRα阳性祖细胞的行为。 
最近，基于荧光激活细胞分选（FACS）的BAs分离方案已经发布12。在该方案中，使用3%牛血清白蛋白（BSA）溶液将BAs与非BAs分离，并通过FACS进一步纯化富集的BAs。该协议的应用受到FACS过程要求的限制，该过程依赖于设备和FACS操作经验。在这项研究中，开发了一种将BA与BAT分离的新协议。通过该方案分离的BA可以直接用于基因和蛋白质表达研究。此外，这项研究的数据表明，BA是PDGF的主要资源。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antigen</td>
			<td>Company</td>
			<td>Catalog</td>
			<td></td>
		</tr>
		<tr>
			<td>PPAR&#947;</td>
			<td>LSBio</td>
			<td>Ls-C368478</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFRa</td>
			<td>Santa Cruz</td>
			<td>sc-398206</td>
			<td></td>
		</tr>
		<tr>
			<td>UCP1</td>
			<td>R&#38;D system</td>
			<td>IC6158P</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical and solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Thermo Fisher Scientific</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Bromo-3-chloropropane</td>
			<td>Sigma-Aldrich</td>
			<td>B62404</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)&#160;</td>
			<td>Goldbio</td>
			<td>A-421-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Bio Basic</td>
			<td>CT1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>IBI Scientific</td>
			<td>IB05040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II, protease</td>
			<td>Sigma-Aldrich</td>
			<td>D5693</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Bio Basic</td>
			<td>EB0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>IBI Scientific</td>
			<td>IB15724</td>
			<td></td>
		</tr>
		<tr>
			<td>LiQuant Universal Green qPCR Master Mix</td>
			<td>LifeSct</td>
			<td>LS01131905Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Boston BioProducts</td>
			<td>P-855</td>
			<td></td>
		</tr>
		<tr>
			<td>OneScrip Plus cDNA Synthesis SuperMix</td>
			<td>ABM</td>
			<td>G454</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep (Iodixanol)</td>
			<td>Cosmo Bio USA</td>
			<td>AXS-1114542</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Caisson Labs</td>
			<td>PBL07</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Caisson Labs</td>
			<td>PBL06</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Boston BioProducts</td>
			<td>P-1435</td>
			<td></td>
		</tr>
		<tr>
			<td>SimplyBlue safe Stain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>75746</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol reagent</td>
			<td>Life technoologies</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gene name (Species)&#160;</td>
			<td>Forward</td>
			<td>Reverse</td>
			<td></td>
		</tr>
		<tr>
			<td>Pdgfra (Mouse)</td>
			<td>CTCAGCTGTCTCCTCACAgG</td>
			<td>CAACGCATCTCAGAGAAAAGG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pdgfa (Mouse)</td>
			<td>TGTGCCCATTCGCAGGAAGAG</td>
			<td>TTGGCCACCTTGACACTGCG</td>
			<td></td>
		</tr>
		<tr>
			<td>36B4(Mouse)</td>
			<td>TGCTGAACATCTCCCCCTTCTC</td>
			<td colspan="2">TCTCCACAGACAATGCCAGGAC</td>
		</tr>
		<tr>
			<td>Ucp1</td>
			<td>ACTGCCACACCTCCAGTCATT</td>
			<td>CTTTGCCTCACTCAGGATTGG</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Application</td>
			<td></td>
		</tr>
		<tr>
			<td>Keyence BZ-X700</td>
			<td>Keyence</td>
			<td colspan="2">Imaging brown adipocytes</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>VWR</td>
			<td>Dissociate BAT</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 6 Flex Real-Time PCR System</td>
			<td>Applied Biosystem</td>
			<td>Quantitative PCR</td>
			<td></td>
		</tr>
		<tr>
			<td>The Odyssey Fc Imaging system</td>
			<td>LI-COR</td>
			<td>Western blot immaging</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating brown adipocytes from murine interscapular brown adipose tissue for gene and protein expression analysis

棕色脂肪组织（BAT）负责哺乳动物的非颤抖产热，棕色脂肪细胞（BA）是BAT的功能单位。BAs含有多房脂滴和丰富的线粒体，它们表达解偶联蛋白1（UCP1）。BA根据其起源分为两种亚型：胚胎衍生的经典BA（cBA）和白色脂肪细胞衍生的BA。由于其密度相对较低，传统的离心方法无法从BAT中分离出来。本研究开发了一种从小鼠中分离BA进行基因和蛋白质表达分析的新方法。在该协议中，用胶原酶和分散酶溶液消化成年小鼠的肩胛间BAT，并用6%碘沙醇溶液富集解离的BA。然后用Trizol试剂裂解分离的BA，以同时分离RNA、DNA和蛋白质。RNA分离后，裂解物的有机相用于蛋白质提取。我们的数据显示，6%碘沙醇溶液有效地富集了BAs，而不会干扰后续的基因和蛋白质表达研究。血小板衍生生长因子（PDGF）是一种调节间充质细胞生长和增殖的生长因子。与棕色脂肪组织相比，分离的BAs具有显着更高的 Pdgfa表达。总之，这种新方法为在单细胞类型水平上研究棕色脂肪细胞的生物学提供了一个平台。

肩胛间BAT用于褐色脂肪细胞分离的制备 棕色脂肪细胞（BA）分离过程如图 1A所示。从制备BAT和消化/分离溶液到获得分离的BA的整个过程大约需要4小时。
在成年小鼠中，丰富的BAT存在于肩胛间区域。这种肩胛间蝙蝠（iBAT）被肌肉层和WAT覆盖（图1B）。在开始消化程序之前，需要去除肌肉层和WAT以产生干净的iBAT（<strong...

Watch this Scientific Journal Video about Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis at JoVE.com

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

本研究描述了一种分离小鼠棕色脂肪细胞用于基因和蛋白质表达分析的新方法。

从小鼠肩胛间棕色脂肪组织中分离棕色脂肪细胞以进行基因和蛋白质表达分析

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs ...

为了研究棕色脂肪组织的生物学方面，必须清除纯化的棕色脂肪大小。目前没有棕色脂肪大小的纯化和质量可用。在这项研究中，我们开发了一个简单的协议来解决这个问题。现在，此协议需要一项技能。与其他方法相比，它需要的研究材料少得多，纯化的棕色脂肪大小可以直接用于基因和蛋白质表达分析。该协议为研究经典棕色脂肪大小的生物学提供了一种新方法。它也可以应用于研究白色脂肪细胞褐变过程。演示该程序的将是我实验室的研究助理阿曼达。首先将装有BAT和消化溶液的烧瓶放在磁力搅拌器上，在35摄氏度的培养箱中以每分钟60转的速度放置30分钟。使用一毫升移液器破坏搅拌棒周围BAT切片的聚集组织团块。消化后，将70微米的过滤器放在干净的50毫升离心管顶部，并通过过滤器移取约4毫升的细胞悬液。然后用四毫升12%碘沙醇溶液洗涤。上下移液，将细胞与碘沙醇溶液混合，然后将细胞混合物转移到两个透明的五毫升聚苯乙烯试管中。将含有细胞混合物的聚苯乙烯管放在冰上一小时，使BA在顶部形成一层。取出20微升分离的BA进行显微镜检查。对于RNA和蛋白质分离，将BA层移液到两个1.7毫升微中心离心管中。然后小心地去除过量的碘溶液，而不会破坏BA层。然后将一毫升TRIzol加入细胞溶液中，以同时分离RNA，DNA和蛋白质，并充分混合以裂解细胞。为了分离相，加入200微升氯仿，并在4摄氏度下以9， 981 G离心10分钟。离心后，使用水相进行RNA分离。将300微升有机相转移到两毫升微量离心管中，并加入750微升100%乙醇。涡旋10秒后，加入200微升1-溴-3-氯丙烷，再次涡旋10秒。在涡旋10秒钟之前加入600微升双蒸水。让混合溶液在室温下静置10分钟。离心后，在4摄氏度下加入9， 981 G10分钟。这些相将被分离，蛋白质相位于中间层。除去顶部的水溶液，并在剩余的溶液中加入一毫升100%乙醇。在4摄氏度下以9，981G离心10分钟后，弃去上清液并用一毫升100%乙醇洗涤沉淀。在 9， 981 G 下在 4 摄氏度下离心 10 分钟。除去上清液后，在室温下风干沉淀10分钟并测量湿沉淀的重量。以每毫克颗粒20微升的比例加入1%SDS溶液。通过将试管放入 55 摄氏度和 11 G 的加热摇床中 5 到 10 分钟，将沉淀完全溶解。在该协议中，含有3%BSA的PBS用于将BA与BAT分开。分离的细胞是覆盆子形的，内部有多房脂滴，并且是tdTom阳性，确认它们是BA。使用改进的GTPC方案从BA中分离蛋白质，然后用SDS-PAGE凝胶进行检查。在BA样品中观察到与BSA相关的显性条带，表明其干扰蛋白质提取。为了避免BSA干扰，使用6%碘氧溶液分离BAs，BAs具有典型的覆盆子形状并含有多房脂滴。从这些BA中提取的蛋白质在SDS-PAGE凝胶中得到很好的分离。发现这些细胞是tdTomato阳性，而从沉淀中回收的细胞是tdTomato阴性，没有明显的脂滴，表明BAs与非脂肪细胞有效分离。在基因表达分析中，分离的BA中UCP1和PDGFA的mRNA水平显着升高。但PDGFRA的mRNA仅在BAT中检测到。在分离的BA中发现富集的UCP1蛋白。EDGFR alpha在BAT中检测到，但在孤立的BA中未检测到。这些结果证实了该方法分离BA的有效性，并证明分离的BA适用于基因和蛋白质表达研究。尝试此方案时，请使用新鲜消化缓冲液解离棕色脂肪组织，并避免在消化期间形成组织团块。这种新方法使得在单个细胞抽样水平上研究棕色脂肪组织的生物学变得容易。通过应用这种方法，可以用纯棕色脂肪细胞精确解剖棕色脂肪细胞基因表达程序进行GN和蛋白质表达研究，而不必担心最初来自非脂肪细胞的污染。

Research

JoVE Journal

Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with ÃƒÅ½Ã‚Â²-arrestin

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

科研

免疫与感染

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation of Adipose Tissue Immune Cells

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...