Name: differentiation and imaging of brown adipocytes from the stromal vascular fraction of interscapular adipose tissue from newborn mice
Uploaded: 2023-02-03T13:10:00
Description: Watch this Scientific Journal Video about Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice at JoVE.com

FONDECYT（1181214和1221146）和Anillos（ACT210039）为VC和博士奖学金提供资金，ANID 21171743 AMF和ANID 21150665 FS。我们感谢亚历杭德罗·穆尼扎加（Alejandro Munizaga）在样品处理方面的帮助和透射电子显微镜的技术建议。插图是使用 BioRender 制作的。

动物程序得到了智利天主教大学的机构动物护理和使用委员会的批准。本研究采用来源于C57BL/6J和129J品系混合背景的P0.5两性新生小鼠。
1. 组织提取
<ol><li>用70%乙醇溶液清洁和消毒工作台，并使用无菌手术材料。</li>
	<li>按照机构批准的方案，用手术剪刀斩首对 P0.5 新生儿幼崽实施安乐死。</li>
	<li>将鼠标置于俯卧位（面朝下），并使用锋利的剪刀在动物背部中?...

<ol>
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本方案是一种简单且可复制的两阶段分化程序（图2），用于产生具有成熟棕色脂肪细胞分子和形态特征的细胞。手术收获 iBAT 是第一个关键步骤，因为组织撕裂严重限制了起始材料的活力。组织处理也是关键，因为不含碎片的均质细胞悬液会大大增加可培养的 SVF 量。在目前的研究中，使用 100 μm 和 40 μm 细胞过滤器进行两次过滤，而其他方案仅使用 100 μm 细胞过滤器 11,12,1...

白色和棕色脂肪组织在其解剖位置、细胞起源、功能、形态和总质量方面有所不同。白色脂肪组织 （WAT） 是人体的主要生理能量储存库，将大量三酰基甘油 （TAG） 储存在高度特化的细胞中，这些细胞具有一个占据其大部分细胞体积的巨大脂滴1。TAG脂肪分解释放游离脂肪酸，游离脂肪酸进入体循环以满足禁食或其他负能量平衡状态期间的能量需求。此外，WAT 分泌蛋白质和脂质产物，分别称为脂肪因子和脂因子，它们具有代谢、免疫和生殖调节功能，从而使 WAT 成为体内最大的内分泌组织2。
棕色脂肪组织 （BAT） 是一个小得多的器官，其主要生理功能是不颤抖产热以防止体温过低。在小鼠和新生儿中，BAT 是位于肩胛间隙的明确器官。成年人缺乏肩胛间 BAT （iBAT）;然而，他们发展出棕色脂肪细胞样细胞簇，这些细胞簇整合在原本主要构成 WAT 的仓库中。这些“棕白”（brite）脂肪细胞与经典的 iBAT 脂肪细胞具有相同的形态和分子特征，但它们具有不同的细胞来源 3,4。
与白色脂肪细胞相比，棕色脂肪细胞具有多个小脂滴和丰富的线粒体5。解偶联蛋白 1（UCP1，也称为热原蛋白）由棕色和棕色脂肪细胞独特表达，并介导线粒体内膜 （IMM） 中的质子泄漏，从而解开 ATP 合成的电子传递并产生热量。BAT 中的非颤抖产热由去甲肾上腺素 （NE） 激活，去甲肾上腺素在冷刺激下从 BAT 的交感神经末梢释放6。NE 与棕色脂肪细胞表面的 β-肾上腺素能受体（主要是 β 3）结合，并触发细胞内 cAMP 介导的信号级联反应。这导致 TAG 脂肪分解、线粒体脂肪酸的 β 氧化以及 UCP1 激活时的热量产生3。棕色脂肪细胞中脂滴和线粒体之间的密切功能关系具有结构上的相似性，例如这些细胞器在大且物理接触非常紧密的区域中的相互作用 7,8。
iBAT具有丰富的血管和交感神经末梢9。这些结构与前脂肪细胞、免疫细胞、成纤维细胞和细胞外基质分子一起组成脂肪基质血管部分 （SVF）10。据报道，许多方案可从前脂肪细胞11、12、13、14、15 产生成熟的脂肪细胞（补充表 1）;然而，它们在组织处理和分化培养基的组成方面表现出极大的变化。本文描述的方案允许有效和可重复地分化棕色脂肪细胞，这些棕色脂肪细胞（1）表达关键的脂肪生成转录因子过氧化物酶体增殖物激活受体γ（PPARγ）和CCAAT /增强子结合蛋白α（C / EBPα），（2）表达成熟的脂肪细胞标志物perilipin1（PLIN1）和簇决定簇36（CD36），（3）积累丰富的脂滴，（4）具有高线粒体质量和高丰度的OXPHOS复合物， （5） 具有产热潜力，由高水平的 UCP1 确定，以及 （6） 具有与成熟棕色脂肪细胞表型相关的线粒体形态变化。该方法用于研究广泛性脂肪营养不良的分子机制 15,16,17。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris/Glycine Buffer</td>
			<td>BioRad</td>
			<td>1610734</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Calbiochem</td>
			<td>410957</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide/Bis Solution, 37.5:1</td>
			<td>BioRad</td>
			<td>1610148</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate&#160;</td>
			<td>SPL Life Science&#160;</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well optical black w/lid cell culture sterile</td>
			<td>Thermo Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuRuler RGB Plus Pre-stained Protein Ladder&#160;</td>
			<td>Maestrogen</td>
			<td>02102-250</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysing buffer&#160;</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate&#160;</td>
			<td>BioRad</td>
			<td>1610700</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>Gibco&#160;</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling</td>
			<td>7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody&#160;&#160;</td>
			<td>Cell Signaling</td>
			<td>7074</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting-grade blocker&#160;</td>
			<td>BioRad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY 493/503&#160;</td>
			<td>Invitrogen</td>
			<td>D3922</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>C/EBP&#945; antibody</td>
			<td>Cell Signaling</td>
			<td>2295</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Calbiochem&#160;</td>
			<td>208291</td>
			<td></td>
		</tr>
		<tr>
			<td>CD36 antibody</td>
			<td>Invitrogen</td>
			<td>PA1-16813</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 100 &#181;m, nylon</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40 &#181;m, nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Gibco</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 Cell Imaging Multimode Reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone&#160;</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, powder</td>
			<td>Gibco</td>
			<td>12500062</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBed-812 EMBEDDING KIT (Epon)</td>
			<td>Electron Microscopy Sciences</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck&#160;</td>
			<td>100983&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from cold water fish skin</td>
			<td>Sigma</td>
			<td>G7041</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Gibco&#160;</td>
			<td>15023-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25% Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>16210</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>Life Technologies</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Phosphatase Inhibitor Cocktail 100X&#160;</td>
			<td>Thermo Scientific</td>
			<td>78427</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail 100X&#160;</td>
			<td>Thermo Scientific</td>
			<td>78429</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Invitrogen</td>
			<td>H1398</td>
			<td></td>
		</tr>
		<tr>
			<td>Immun-Blot PVDF Membrane&#160;</td>
			<td>BioRad</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin&#160;</td>
			<td>Sigma&#160;</td>
			<td>I7378&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma</td>
			<td>I3536</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Calbiochem&#160;</td>
			<td>529552</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Calbiochem</td>
			<td>529568</td>
			<td></td>
		</tr>
		<tr>
			<td>KHCO3</td>
			<td>Sigma</td>
			<td>60339</td>
			<td></td>
		</tr>
		<tr>
			<td>Lane Marker Reducing Sample Buffer&#160;</td>
			<td>Thermo Scientific</td>
			<td>39000</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ water sterile&#160;</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitoprofile Total OXPHOS Rodent WB antibody Cocktail</td>
			<td>Abcam&#160;</td>
			<td>MS604</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merck&#160;</td>
			<td>1064041000</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur&#160;10x PBS Liquid Concentrate</td>
			<td>Calbiochem</td>
			<td>6505-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>19100</td>
			<td></td>
		</tr>
		<tr>
			<td>Perilipin 1 antibody</td>
			<td>Cell Signaling</td>
			<td>9349</td>
			<td></td>
		</tr>
		<tr>
			<td>PPAR&#947; (81B8) antibody</td>
			<td>Cell Signaling</td>
			<td>2443</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer lysis&#160;</td>
			<td>Thermo Scientific</td>
			<td>89901</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Merck</td>
			<td>557366</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacpdylate</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate&#160;</td>
			<td>BioRad</td>
			<td>1610301</td>
			<td></td>
		</tr>
		<tr>
			<td>T3</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Talos F200C G2</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED&#160;</td>
			<td>BioRad</td>
			<td>1610800</td>
			<td></td>
		</tr>
		<tr>
			<td>TOM20 antibody</td>
			<td>Cell Signaling</td>
			<td>42406</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffered Saline (TBS-10X)</td>
			<td>Cell Signaling</td>
			<td>12498</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20, Molecular Biology Grade</td>
			<td>Promega&#160;</td>
			<td>H5152</td>
			<td></td>
		</tr>
		<tr>
			<td>UCP1 antibody</td>
			<td>Cell Signaling</td>
			<td>14670</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracut R</td>
			<td>Leica&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
		<tr>
			<td>Westar Sun&#160;</td>
			<td>Cyanagen</td>
			<td>XLS063</td>
			<td></td>
		</tr>
		<tr>
			<td>Westar Supernova&#160;</td>
			<td>Cyanagen</td>
			<td>XLS3</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation and imaging of brown adipocytes from the stromal vascular fraction of interscapular adipose tissue from newborn mice

棕色脂肪组织（BAT）仅存在于哺乳动物中，具有产热功能。棕色脂肪细胞的特征是多房细胞质，具有多个脂滴、中心核、线粒体含量高和解偶联蛋白 1 （UCP1） 的表达。BAT 已被提议作为肥胖及其相关代谢紊乱的潜在治疗靶点，因为它能够将代谢能作为热量耗散。为了研究BAT的功能和调控，棕色脂肪细胞培养是必不可少的。本方案优化了从新生小鼠培养棕色脂肪细胞的组织处理和细胞分化。此外，还显示了使用共聚焦免疫荧光和透射电子显微镜对分化脂肪细胞进行成像的程序。在与本文所述技术分化的棕色脂肪细胞中，保留了经典 BAT 的主要定义特征，包括高 UCP1 水平、增加的线粒体质量以及脂滴和线粒体之间非常紧密的物理接触，使该方法成为 BAT 研究的宝贵工具。

脂肪生成受转录因子网络的调节，这些转录因子负责诱导棕色脂肪细胞形成和功能的关键蛋白的表达22，包括经典的脂肪生成调节因子，如 PPARγ 和 C/EBPα 23,24,25，以及成熟脂肪细胞的标志物 26,27.通过测试允许获取产热棕色脂肪表型的不同浓度的罗格列酮，本文描述...

Watch this Scientific Journal Video about Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice at JoVE.com

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

前脂肪细胞是从新生小鼠肩胛间棕色脂肪组织的基质血管部分中分离出来的，并分化成积累脂滴、表达分子标记物并显示成熟棕色脂肪细胞线粒体形态的细胞。通过免疫荧光和透射电子显微镜进一步分析这些细胞。

新生小鼠肩胛间脂肪组织基质血管部分棕色脂肪细胞的分化和成像

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

该方案允许有效和可重复地分化表达关键脂肪生成转录因子，成熟脂肪细胞标志物的棕色脂肪细胞，并具有成熟棕色脂肪细胞的产热电位和形态表型。这是一种简单且可复制的两阶段分化程序，可从新生小鼠的肩胛间棕色脂肪组织中获得具有成熟棕色脂肪细胞特征的细胞。该协议允许研究棕色脂肪组织的激活机制作为治疗肥胖的靶点。它一直是确定脂肪营养不良病理生理学机制的模型。首次执行这种技术预计会很有挑战性。必须有一个详细的协议并强调关键点，以获得成功的结果。首先，将安乐死的小鼠置于俯卧位，并使用锋利的剪刀在动物背部的中部做一个一厘米长的皮肤切口。小心地去除皮肤，使用小剪刀通过手术将iBAT从胴体上分离。使用曲线尖钳收获两个 iBAT 裂片，以独立移除裂片。在室温下将两个裂片放入装有 1.5 毫升无菌 PBS 的塑料管中。将 iBAT 组织在 37 摄氏度的胶原酶 2 型消化缓冲液中孵育 45 分钟，以 800 RPM 的速度连续轻轻摇动。通过使用P1000微量移液器和过滤器吸头上下移液，机械地破坏胶原酶二型消化缓冲液中的组织悬浮液。为每个样品使用新的吸头。将解离的组织通过单独的 100 微米细胞过滤器放入新的 1.5 毫升管中。向过滤的组织样本中加入 500 微升 ACK 缓冲液。将试管倒置四到五次，并在室温下孵育四分钟。然后使用 P1000 微量移液器和过滤吸头将悬浮液通过 40 微米细胞过滤器放入新的 1.5 毫升管中。离心使颗粒重悬，如文本手稿中所述。然后通过向每个孔中加入一毫升悬浮液，将每个样品接种到 24 孔培养板的六个孔中。未分化的前脂肪细胞表现出非常低或无法检测到的 ppar-γ、C/ebp-α、perilipin 1 和 CD36 水平。相比之下，这些标志物在分化的第七天明显更高。棕色前脂肪细胞中多个脂滴的积累与先前发表的结果一致。在分化第 7 天，观察到线粒体质量标志物 TOM 20 和氧化磷酸化复合物蛋白水平显着增加。UCP1 是该细胞类型的真正标志物，在未分化的前脂肪细胞中未检测到，而在分化的第 7 天，它在成熟的棕色脂肪细胞中大量表达。线粒体从第0天的细长管状进化到第七天的圆形或豆状。线粒体内部结构也通过脂肪生成而改变，导致平行堆积的 crystae 密度更高。重要的是，线粒体与分化的棕色脂肪细胞中的脂滴密切相关，因此即使使用高分辨率透射电子显微镜，也无法检测到线粒体外膜与脂滴表面之间的明显距离。肩胛间脂肪组织的手术切除至关重要。组织数量低下限制了将要分化的细胞的可用性。这种成像技术有助于在体外表征分化的棕色脂肪细胞的形态。可以进行线粒体功能、β 雄激素激活和自噬抑制的检测。

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

科研

生物学

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

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

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 ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

使用[18F]FDG微PET / MR成像对小鼠中的棕色和米色脂肪组织进行可视化和定量

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...