这项工作得到了国家自然科学基金（82130012和81830010）和上海市胸科医院基础研究培育项目（批准号:2022YNJCQ03）的支持。

动物用药方案经上海交通大学医学院附属上海胸科医院机构动物护理与使用委员会批准（批准文号:KS23010），符合相关伦理规范。4-8周龄的雄性和雌性C57BL / 6小鼠是该实验的首选。
1. 手术工具、缓冲液和培养基的制备
<ol><li>将手术工具（例如手术剪刀和标准镊子）在121°C高压灭菌30分钟。用75%酒精对显微外科器械进行消毒。</li>
	<li>制备补充有 1% v/v 青霉素 - 链霉素的无菌磷酸盐缓冲盐水 （PBS） 和另外 10 mL 补充有 10% v/v 青霉素 - 链霉素的 PBS。 注意:在以下各节中，如果没有特别提及，PBS 是指常规无菌 PBS。</li>
	<li>制备克雷布斯林格HEPES BSA缓冲液:1%牛血清白蛋白（BSA），20mM HEPES溶解在克雷布斯林格溶液中。</li>
	<li>制备新鲜消化溶液:1型胶原酶（1mg / mL）和分散酶II（4mg / mL）溶解在Krebs Ringer HEPES BSA缓冲液中。使用0.2μm过滤器对溶液进行灭菌。</li>
	<li>准备胶原蛋白包被的 12 孔板。<ol><li>用无菌去离子水稀释胶原蛋白溶液（1mg / mL）至40μg/ mL的浓度。在每个孔的表面加入 1 mL 稀释的胶原蛋白溶液，以达到 6-10 μg/cm 2 的浓度。将涂层在室温下孵育1小时。</li>
		<li>取出剩余的溶液，用 1 mL PBS 冲洗每个孔 2 次。立即使用板，或在II级层流罩中风干板，并将涂层板储存在2-8°C。 储存时，用封口膜密封涂层板。</li>
	</ol></li>
	<li>制备培养基:高葡萄糖 Dulbecco 改良的 Eagles 培养基 （DMEM）、10% 胎牛血清 （FBS）、1% v/v 青霉素 - 链霉素。在4°C下保持长达2周。</li>
	<li>制备脂肪生成诱导培养基:高葡萄糖DMEM，10%FBS，1%v / v青霉素 - 链霉素，1nM三碘甲状腺原氨酸，1μM罗格列酮，1μM胰岛素，0.5mM3-异丁基-1-甲基黄嘌呤（IBMX）和1μM地塞米松。在4°C下保持长达2周。</li>
	<li>准备维持培养基:高葡萄糖DMEM，10%FBS，1%v / v青霉素 - 链霉素，1nM三碘甲状腺原氨酸，1μM罗格列酮和1μM胰岛素。在4°C下保持长达2周。</li>
</ol>2. 血管周围脂肪组织（PVAT）的解剖和分离
<ol><li>通过在2%异氟烷麻醉下或二氧化碳过量的颈椎脱位对小鼠实施安乐死。喷洒75%酒精进行皮肤消毒。</li>
	<li>将鼠标置于仰卧位（面朝上）。</li>
	<li>提起皮肤，做一个小切口，用手术剪刀沿腹侧中线从骨盆到颈部钝性地分离皮肤和腹部肌肉。通过沿中线两侧切割横膈膜和肋骨来暴露心脏和肺部。</li>
	<li>小心切除肝脏、脾脏、肠道和肾脏。避免切割肠壁。</li>
	<li>切除肺和食道。</li>
	<li>一只手用镊子轻轻提起心脏，另一只手用手术剪刀将主动脉与脊柱分开。然后，将心脏和主动脉置于含有1%v / v青霉素 - 链霉素的PBS的60mm培养皿中。</li>
	<li>从酒精中取出显微外科镊子和显微外科剪刀，然后用 25 mL PBS 冲洗以除去多余的酒精。在立体显微镜下，从心脏和主动脉中取出胸腺和其他组织，然后取出心脏，将主动脉与PVAT一起留下。</li>
	<li>将带有PVAT的主动脉转移到含有1%v / v青霉素 - 链霉素的PBS的干净60mm培养皿中。</li>
	<li>使用显微手术镊子剥离PVAT，一对镊子固定主动脉，另一对拉出脂肪组织。尽可能切除脉管组织，同时尽量减少对血管周围脂肪组织的损伤。</li>
	<li>将胸主动脉周围的 PVAT 收集到含有补充有 1% v/v 青霉素 - 链霉素的高葡萄糖 DMEM 的 2 mL 微量离心管中，置于冰上。</li>
</ol>3. 基质血管分数（SVF）的分离
<ol><li>用含有10%v / v青霉素 - 链霉素的PBS和含有1%v / v青霉素 - 链霉素的PBS依次冲洗收集的组织。 注意:从此步骤开始，所有实验都必须在无菌条件下在II级层流罩中进行。</li>
	<li>将组织转移到无菌的 60 mm 培养皿中，加入 200 μL 消化溶液，并使用无菌剪刀将它们切成 1 mm3 块。</li>
	<li>使用塑料移液器吸头将混合物转移到 15 mL 离心管中，末端通过无菌剪刀切开，并加入 6 mL 消化溶液以开始消化反应。 注:一个消化反应（3mL消化溶液）足以用于来自六只小鼠的PVAT仓库。</li>
	<li>将组织在37°C下在带有轨道振荡器（150rpm频率用于有效混合）的培养箱中孵育30-45分钟。将试管平放在架子上，使试管水平摇晃。每 5-10 分钟用手剧烈上下摇晃 10 秒。 注意:当看到均匀的淡黄色消化液而没有组织碎片时，可以停止消化。</li>
	<li>通过 70 μm 细胞过滤器将消化的组织过滤到 50 mL 离心管中。用等体积的培养基冲洗细胞过滤器，以最大限度地提高细胞产量并停止消化。</li>
	<li>将滤液转移到新的 15 mL 离心管中，并以 1,800 × g 离心 10 分钟。倒置试管以弃去上清液，并将沉淀重悬于 5 mL PBS 中。以1,800× g 离心5分钟。</li>
	<li>通过倒置管弃去上清液，并将沉淀重悬于适当体积的培养基中。 注:从每六只小鼠收集的细胞沉淀重悬于1mL培养基中，并接种到12孔板的一个孔中。</li>
	<li>将细胞接种到胶原包被的12孔板中，并在37°C下在含有5%CO2 的潮湿气氛中孵育过夜。</li>
	<li>第二天，吸出培养基，用含有1%v / v青霉素 - 链霉素的预热（37°C）PBS洗涤细胞以除去细胞碎片和红细胞，并加入1mL新鲜培养基。</li>
</ol>4. 主动脉周围脂肪组织中SVF衍生的前脂肪细胞的成脂诱导
<ol><li>每隔一天更换一次培养基，直到细胞达到~60-70%汇合。 注意:细胞通常需要 3-4 天才能达到 60-70% 的汇合度。</li>
	<li>当细胞达到60-70%汇合时，吸出培养基并用棕色脂肪诱导培养基代替。将诱导脂肪分化的日期视为分化的第 0 天。</li>
	<li>72小时后（分化的第3天），用维持培养基刷新培养基。每2天更换一次维持培养基，直到细胞用于实验。</li>
	<li>以适当的方式分析脂肪生成细胞，例如油红O染色14 和蛋白质印迹15。</li>
</ol>

培养从小鼠血管周围脂肪组织中分离的前脂肪细胞

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作者没有财务或其他利益冲突需要声明。

我们提出了一种实用可行的方法，用于从小鼠主动脉周围脂肪组织中分离和诱导SVF来源的前脂肪细胞。该协议的优点是简单且经济。足够数量的小鼠对于成功分离至关重要，因为组织不足会导致 SVF 密度低和生长状态不佳，最终影响脂肪生成效率。此外，小鼠年龄是一个需要考虑的重要因素，因为SVF的成脂潜力随着年龄的增长而降低。快速、仔细地分离PVAT，同时最大限度地减少脉管系统的污染至?...

血管周围脂肪组织 （PVAT） 是一种由成熟脂肪细胞和基质血管部分 （SVF） 混合物组成的血管周围结构，被认为通过其分泌组旁分泌与相邻血管壁相互作用1。作为血管稳态的关键调节因子，PVAT 功能障碍与心血管疾病的发病机制有关 2,3,4。脂肪细胞组织的 SVF 由几个预期的细胞群组成，包括内皮细胞、免疫细胞、间皮细胞、神经元细胞以及脂肪干细胞和祖细胞 （ASPC）5,6。众所周知，存在于脂肪组织SVF中的ASPC可以产生成熟的脂肪细胞5。SVF 被推断为 PVAT 中成熟脂肪细胞的关键来源。多项研究表明，PVAT-SVF 可以在特定的诱导条件下分化为成熟的脂肪细胞 6,7,8。
目前，从脂肪组织中分离SVF有两种分离系统，一种是酶消化，另一种是非酶9。酶法通常可提高有核祖细胞的产量10。迄今为止，SVF 在促进伤口愈合、泌尿生殖系统和心血管疾病中的血管再生和新生血管形成方面的益处已得到广泛证明11，尤其是在皮肤病学和整形外科领域12,13。然而，PVAT衍生SVF的临床应用前景尚未得到很好的探索，这可能是由于缺乏标准化的SVF与PVAT分离方法。该协议的目的是建立一种标准化方法，用于从胸主动脉周围的小鼠PVAT中分离，培养和脂肪生成诱导SVF衍生的前脂肪细胞，从而能够进一步研究PVAT功能。该方案优化了组织加工和细胞分化技术，用于培养从年轻小鼠获得的主动脉周围脂肪细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>0.2 &#956;m syringe filters</td>
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			<td>4612</td>
			<td></td>
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			<td>12-well plate&#160;</td>
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			<td>15 mL centrifuge tube</td>
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			<td>3,3&#39;,5-triiodo-L-thyronine (T3)</td>
			<td>Sigma-Aldrich</td>
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			<td>50 mL centrifuge tube</td>
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			<td>anti-UCP1</td>
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			<td>ab10983</td>
			<td>1:1,000 working concentration</td>
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			<td>anti-&#945;-Actinin</td>
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			<td>6487</td>
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			<td>BSA</td>
			<td>Beyotime</td>
			<td>ST023-200g</td>
			<td>1%</td>
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			<td>C57BL/6 mice aged 4-8 weeks of both sexes</td>
			<td>Shanghai Model Organisms Center, Inc.</td>
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			<td>Cell Strainer 70 &#181;m, nylon</td>
			<td>Falcon</td>
			<td>352350</td>
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			<td>Collagen from calf skin</td>
			<td>Sigma-Aldrich</td>
			<td>C8919</td>
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			<td>Collagenase, Type 1</td>
			<td>Worthington</td>
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			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td>1 &#956;M</td>
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			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>D4693-1G</td>
			<td>4 mg/mL</td>
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			<td>Fetal bovine serum&#160;</td>
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			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-25G</td>
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			<td>High glucose DMEM</td>
			<td>Hyclone</td>
			<td>SH30022.01</td>
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			<td>IBMX&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td>0.5 mM</td>
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			<td>Incubator with orbital shaker</td>
			<td>Shanghai longyue Instrument Eruipment Co.,Ltd.</td>
			<td>LYZ-103B</td>
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			<td>Insulin (cattle)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>11070-73-8</td>
			<td>1 &#956;M</td>
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		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Ringer&#39;s Solution</td>
			<td>Pricella&#160;</td>
			<td>PB180347</td>
			<td>protect from light&#160;</td>
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		<tr>
			<td>Microsurgical forceps</td>
			<td>Beyotime</td>
			<td>FS233</td>
			<td></td>
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		<tr>
			<td>Microsurgical scissor</td>
			<td>Beyotime</td>
			<td>FS217</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O&#160;</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd</td>
			<td>A600395-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd</td>
			<td>B548117-0500</td>
			<td></td>
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			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
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		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>115-035-146</td>
			<td>1:5,000 working concentration</td>
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		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-035-144</td>
			<td>1:5,000 working concentration</td>
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		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>Standard forceps</td>
			<td>Beyotime</td>
			<td>FS225</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Beyotime</td>
			<td>FS001</td>
			<td></td>
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isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue

血管周围脂肪组织 （PVAT） 是一种围绕血管的脂肪组织库，表现出白色、米色和棕色脂肪细胞的表型。最近的发现揭示了PVAT在调节血管稳态和参与心血管疾病发病机制中的核心作用。全面了解PVAT特性和调控对于未来疗法的开发非常重要。主动脉周围脂肪细胞的原代培养对于研究PVAT功能以及主动脉周围脂肪细胞与血管细胞之间的串扰很有价值。本文提出了一种经济可行的方案，用于从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞，可用于 体外模拟脂肪生成或脂肪生成。该方案概述了用于培养年轻小鼠主动脉周围脂肪细胞的组织处理和细胞分化。该协议将为研究PVAT功能提供工作台侧的技术基石。

使用上述方案，我们仔细分离小鼠胸主动脉周围的PVAT（图1A-D）。使用无菌剪刀将PVAT洗涤并切碎成小块（图1E，F）后，将组织碎片在含有1型胶原酶（1mg / mL）和分散酶II（4mg / mL）的消化溶液中消化，并在37°C下在振荡器上孵育30-45分钟（图1G）。将消化的组织通过70μm细胞过滤器过滤到50mL离心...

Watch this Scientific Journal Video about The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue at JoVE.com

作者聚焦:从小鼠血管周围脂肪组织中分离、培养和诱导 SVF 衍生的前脂肪细胞的成脂率 

在这里，我们描述了从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞，从而可以研究血管周围脂肪组织功能及其与血管细胞的关系。

从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

我们的目标是建立一种标准化的方法，用于从大规模 PVAT 中分离和诱导基质血管部分来源的前脂肪细胞的脂肪前细胞。该协议使我们能够在体外对PVAT功能以及主动脉周围脂肪细胞和血管细胞之间的通信进行进一步研究。我们目前在实验中面临的主要挑战包括PVAT的可用性受到限制，以及与使用永生化前脂肪细胞相比，需要额外的人力和物力资源。我们的方案允许研究血管周围脂肪组织功能及其与血管细胞的关系。我们还为研究PVAT环和基因以及异质性提供了基础，并为探索PVAT分化和体外脂肪生成的重要调控因子提供了平台。

isolation-culture-adipogenic-induction-stromal-vascular-fraction

Research

JoVE Journal

Biology

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

1.9K 次观看.  Shanghai Jiao Tong University School of Medicine. 我们的目标是建立一种标准化的方法，用于从大规模 PVAT 中分离和诱导基质血管部分来源的前脂肪细胞的脂肪前细胞。该协议使我们能够在体外对PVAT功能以及主动脉周围脂肪细胞和血管细胞之间的通信进行进一步研究。我们目前在实验中面临的主要挑战包括PVAT的可用性受到限制，以及与使用永生化前脂肪细胞相比，需要额外的人力和物力资源。我们的方案允许研究血?...

视频: 作者聚焦:从小鼠血管周围脂肪组织中分离、培养和诱导 SVF 衍生的前脂肪细胞的成脂率 

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

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

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 ...

科研

生物学

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

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

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

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 ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

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

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

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 ...

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

测量体外分化的离体小鼠脂肪组织和原代前脂肪细胞的脂肪分解率

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

脂肪组织来源的干细胞分离和表征

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

作者聚焦:分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

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

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

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  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

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 ...