我们确认梅奥诊所显微镜细胞分析核心流细胞学设施，以协助FACS分拣。

所有动物实验程序均经梅奥诊所机构动物护理和使用委员会批准。
1. 解决方案准备 
<ol><li>通过溶解拼贴酶 II 2 g 在 100 mL 汉克斯的平衡盐溶液 （HBSS） 中准备拼贴酶 2% （w/v） 溶液。每次使用均报价200μL。</li>
	<li>混合84毫升F-12介质、15毫升马血清和1毫升青霉素/链霉素，制备中和介质。</li>
	<li>通过溶解 500 毫克牛血清白蛋白 （BSA） 和 400 μL 的 0.5 M EDTA 在 100 mL 杜尔贝科的磷酸盐缓冲盐水 （DPBS） 中准备流细胞学缓冲。</li>
</ol>2. 表皮脂肪垫解剖和组织分离
<ol><li>人道安乐死（异体/颈椎错位）一只雄性小鼠。在此协议中，使用了一个四个月大的 FVB 鼠标。</li>
	<li>在腹部涂抹/喷洒 70% 乙醇，使用干净的剪刀和钳子暴露下腹腔。</li>
	<li>定位表皮脂肪垫（近似/附着在睾丸上）。用钝力握住睾丸和表皮脂肪垫之间的交界处，轻轻拉动以解放表皮脂肪垫。</li>
	<li>使用剪刀切割睾丸，在 5 mL 的 HBSS 中孵育表皮脂肪垫，在室温下在 50 mL 圆锥管中补充 3% BSA 15 分钟。</li>
	<li>离心机在150 x g 7分钟在4°C。</li>
	<li>从 50 mL 圆锥管中取出漂浮的表皮脂肪垫，用干净的剪刀细碎组织。</li>
	<li>在 13 mL 培养管中将 200μL 拼贴酶 2% 溶液添加到 3.8 mL 的 HBSS 中。加入切碎的组织，在 5 rpm 旋转孵化器中孵化，在 37 °C 下为 1 小时。 37 °C 的细胞培养孵化器可以偶尔搅拌使用，而不是旋转孵化器。</li>
	<li>将整个内容传输到 50 mL 圆锥管中，并添加 10 mL 的中和介质。通过倒置管 2-3 次轻轻混合。</li>
	<li>再准备一个装有70μm细胞过滤器的50mL圆锥管。使用细胞过滤器将消化的组织过滤到新管中。</li>
	<li>在 4 °C 下将流经传输到 15 mL 圆锥管和离心机，在 350 x g 下传输 10 分钟。</li>
	<li>小心地删除超纳坦和重新悬浮的细胞颗粒与5 mL的DPBS。离心机在350 x g 10分钟在4°C。</li>
	<li>小心地删除超纳坦，并添加 50 μL 的流细胞学缓冲区。轻轻吹笛，将细胞放在冰上，混合好。由于细胞颗粒的体积和少量的残余超常剂，流细胞学缓冲区中的细胞总体积将大于 50 μL（约 100 μL）。</li>
</ol>3. 抗体标记和荧光激活细胞分拣 （FACS）
<ol><li>将细胞与温和的管道混合后，将54μL的细胞悬架转移到1.7 mL微中心管中。加入 6 μL 的 FcR 阻塞试剂，轻轻吹笛混合。在4°C下孵化10分钟。服用 54 μL 后保存任何剩余的细胞，并将它们保存在冰中，作为第 3.5 步和第 3.6 步的未染色控制。</li>
	<li>在步骤 3.1 的孵化过程中，通过在微中心管中结合 11 μL 的抗 Sca1-APC、抗 Ter119-FITC、抗 CD31-FITC 和抗 CD45-FITC 抗体，制备抗体鸡尾酒。通过温和的管道混合好，并保持在冰上保护免受光线。</li>
	<li>用 FcR 阻断试剂孵育 10 分钟后，加入 40 μL 的抗体鸡尾酒，通过温和的管道混合。在4°C下孵化10分钟。</li>
	<li>添加 500 μL 的流细胞学缓冲区，并辅以 1 μg/mL 的 DAPI 进入染色细胞。使用带细胞滤光器扣盖的 5 mL 试管，通过温和的管道和过滤细胞很好地混合。将细胞放在冰上。</li>
	<li>在步骤 3.1 中将 500μL 的流细胞学缓冲器添加到剩余的细胞中，并通过温和的管道很好地混合。使用带细胞滤光片捕捉帽的 5 mL 试管过滤细胞。将这些细胞保持在冰上，并在下一步用作未染色的控制。</li>
	<li>将染色细胞和未染色控制带到 FACS 分析或分拣仪器中。<ol>
<li>识别和隔离 APC+/ FITC-/ DAPI-人口与图 1中显示的量子策略。这些细胞代表可行的Sca1+/Ter119-/CD31-/CD45-细胞。使用前向 （FSC） 和侧散射 （SSC） 图耗尽碎片和细胞聚合物。然后，DAPI-人口是封闭的，然后是APC+/FITC-人口的门控化。应使用未染色的控件来帮助设置界点参数。</li>
		<li>在 1.5 mL 微中心管中将细胞收集到 500μL 的流动细胞学缓冲液中。今后，所有程序都应在无菌条件下进行，以尽量减少污染。大约50，000-100，000个细胞从一只小鼠身上收集。</li>
	</ol>
</li>
	<li>使用血细胞计计算细胞以评估细胞存活率。由于细胞聚合可以干扰进一步的下游分析，也评估细胞聚合的存在，以确保这些聚合物的最低存在（+lt;5%的可行细胞数）。</li>
</ol>

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作者没有什么可透露的。

单细胞RNA测序（scRNA-seq）作为同时研究单细胞水平不同细胞群的有力工具，正迅速获得牵引力。由于样品制备成本高，吞吐量测序高，因此必须优化蜂窝输入（高可行性和纯度），以提高实验成功的可能性。一些细胞制备方案依赖于使用低旋转洗涤和基于柱的分离去除死细胞和碎片，而没有FACS分类14。然而，许多这些方法大大减少了存活的恢复细胞的数量，在许多情况下，死细胞并没有完全被切除14个。该协议概述了一个简单的方法来分离高度可行的Sca1+ 脂肪祖细胞含有最小的细胞碎片，细胞双胞胎和死细胞。虽然使用此协议分离的细胞具有很高的生存能力，但我们仍然建议将细胞分离和进一步下游分析之间的时间最小化，以保持较高的生存能力。此外，成功的抗体标记对于分离具有高纯度的Sca1+脂肪祖细胞至关重要。因此，强烈建议确定每个抗体的最佳浓度，并可以相应地调整第3.2步中的抗体稀释。
此基于流细胞学的协议是适应定制，这取决于个人对特定脂肪祖细胞子群的兴趣。在这里，替代抗体组合可用于识别和隔离感兴趣的人口。事实上，由于广泛的 APC 标记多样性，多个实验室使用 scRNA-seq 报告使用其他表面标记分离细胞来研究 APC。例如，PDGFRA+/CD44+细胞，CD31-/CD45-/Ter119-/CD29+/CD34+/Sca1+细胞，Pdgfrb+细胞已被 FACS 分离，用于下游 APC scRNA-seq 分析8、9、11。CD142+亚人口也已的特点，并显示表现出有限的致波分化能力和抑制脂肪生成10，11的能力。除Sca1外，最近还利用CD55、CD81和CD9的抗体组合，对ASC和前细胞亚群（使用本协议）进行前瞻性分离，研究亚人口分子和功能异质性7。此外，由于其他脂肪垫（包括脂肪垫和棕色脂肪组织）的脂肪祖细胞已经像现行协议15、16、17一样，使用拼贴酶分离分离，本协议可能适用于从其他脂肪组织中分离脂肪祖细胞。
目前方法的一个警告是，它是为孤立不成熟的APC而量身定做的。虽然本协议产生高生存能力的ACC，但其他脂肪组织常驻细胞类型（即免疫细胞、成纤维细胞、内皮细胞等）的分离效果和效率尚不清楚。重要的是，此协议不适合分离成熟的脂肪细胞。由于脂肪细胞的细胞大小大且脆弱，因此很难用FACS分离。最近，FACS的成熟脂肪细胞的分离报告使用大喷嘴大小（150μm）和低护套压力（6 psi）与特定的FSC/SSC门治策略18.未来的研究或许可以将这种脂肪细胞分离协议与此处描述的协议合并，以建立一个更全面的管道来分离和研究脂肪组织内的不同细胞类型。

脂肪组织在能量代谢中起着关键作用。多余的能量以脂质的形式储存，脂肪组织能够根据营养状况和能量需求显著扩张或缩水。脂肪组织的扩张可能是由于脂肪细胞大小（肥大）和/或脂肪细胞数（增生）的增加造成的:后一过程受脂肪祖细胞1、2的增殖和分化的严格调控。在肥胖期间，脂肪组织过度扩张，组织功能障碍（包括缺氧、炎症和胰岛素抵抗）往往发展为3、4。这些并发症是许多慢性疾病的危险因素，包括高血压、糖尿病、心血管疾病、中风和癌症。因此，限制不受控制的脂肪组织扩张和缓解脂肪组织病理学是生物医学研究的首要任务。在脂肪组织扩张过程中，驻地脂肪组织衍生的干细胞（ASCs）会增殖并按顺序分化成前细胞（承诺的祖细胞），然后发育成成熟的脂肪细胞6。最近的单细胞RNA测序（scRNA-seq）研究表明，这些脂肪祖细胞（APC）种群（ASC和前细胞）表现出大量的分子和功能异质性7，8，9，10，11，12。例如，与前极细胞7相比，ASC在表现出低致病性分化能力的同时，也表现出更高的增殖和扩张能力。在ASC和前细胞群中报告了进一步的分子差异，尽管这些差异的功能相关性仍不清楚7。这些数据共同突出了脂肪祖细胞库的复杂性，并强调了开发和标准化工具以更好地了解和操纵这些关键细胞群的必要性。
该协议详细说明了高存活率Sca1+脂肪祖细胞群与小鼠表皮脂肪垫的分离，这些脂肪垫适合敏感的下游分析，包括单细胞研究（scRNA测序）和细胞培养。表皮脂肪垫的分离和分离按照先前描述的7，13进行，稍作修改，改善孤立的APC的可行性。简言之，表皮脂肪垫上的分离细胞沾染了对Sca1的抗体，Sca1是ASC和前细胞6、7和其他血统（林）标记的标记:Ter119（红细胞）、CD31（内皮细胞）和CD45（白细胞）。可行的 Sca1+/ Ter119-/CD31-/CD45-/DAPI-细胞然后通过荧光激活细胞排序 （FACS） 进行排序。重要的是，在最近的单细胞RNA测序研究中，成功分离和分析可行的Sca1+/Lin-脂肪祖细胞，确定了ASC和前细胞7中的功能异质亚群，验证了这一协议。

<table border="0" cellpadding="0" cellspacing="0" style="width:799px;" width="799">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1.7 mL microcentrifuge tube</td>
			<td style="width:181px;">VWR</td>
			<td style="width:119px;">87003-294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">13 mL culture tube</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-809-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">15 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">188 271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5 mL test tube with cell strainer snap cap</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">08-771-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">50 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">70 &#181;m cell strainer</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD31-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD45-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Sca1-APC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Ter119-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-112-908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BSA</td>
			<td style="width:181px;">Gold Biotechnology</td>
			<td style="width:119px;">A-420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Collagenase type II</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17101-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DAPI</td>
			<td style="width:181px;">Thermo Fisher</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">F-12 medium</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FcR blocking reagent</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hanks&#39; balanced salt solution (HBSS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Horse serum</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Penicillin-streptomycin</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Propidium iodide solution</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-233</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of adipose progenitor cells from mouse epididymal adipose tissues

肥胖症和代谢性疾病，如糖尿病、心脏病和癌症，都与戏剧性的脂肪组织重塑有关。组织驻地脂肪祖细胞（APC）在脂肪组织平衡中起着关键作用，有助于组织病理学。单细胞分析技术（包括单细胞RNA测序和单细胞蛋白组学）的日益使用，通过允许在人口或组织范围内对单个细胞表达变化进行前所未有的解决，正在改变干细胞/祖细胞领域。在本文中，我们提供了详细的协议，以解剖小鼠表皮脂肪脂肪组织，分离单个脂肪组织衍生细胞，并执行荧光激活细胞排序（FACS），以丰富可行的Sca1+/CD31-/CD45-/Ter119- APCs。这些协议将允许调查人员准备适合下游分析的高质量APC，如单细胞RNA测序。

这个实验使用了四个月大的雄性FVB小鼠。在排除使用FSC/SSC地块的碎片和双胞胎后，对可行的细胞（DAPI- 人口）进行了封闭，随后选择了APC+/FITC- 人口（图1）。DAPI、APC 和 FITC 门是根据未染色的控制绘制的。在 图 1中显示的量化策略。
经过1小时的排序，通过流细胞学分析（图2，3）对隔离质量进行了定量评价。细胞被碘化铀溶液（1:100）染色，用于生存性染色。使用相同的量子进行分拣，细胞保持了较高的生存能力和纯度:92.6±2.2%的单细胞（图2中的第三面板）、86.4±3.0%的可行性（PI-细胞）和86.0±2.8%的APC+/FITC-细胞（n= 4，平均±标准偏差）。这些百分比被定义为每个封闭人口（单细胞、PI-细胞和 PI-/APC+/FITC-人口百分比，分别在图 2中）相对于总细胞数。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61694/61694fig1v2.jpg"> 图1:通过FACS分离可行的Sca1+ 脂肪祖细胞单细胞。 在染色细胞和未染色控制下显示盖廷策略。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61694/61694fig1v2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61694/61694fig2v2.jpg"> 图2:代表性流细胞学分析，以评估隔离后细胞的生存能力。 使用碘化铀进行活性染色。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61694/61694fig2v2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61694/61694fig3.jpg"> 图3:流细胞学分析中细胞1小时后分离百分比的量化。 单细胞、活细胞和 APC+/FITC- 细胞的百分比被量化，以评估细胞分离后的纯度和可行性。显示的数据表示平均±标准偏差。n=4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61694/61694fig3large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

我们提出了一个简单的方法，利用荧光激活细胞分拣从小鼠表皮脂肪垫中分离出高度可行的青春期祖细胞。

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

preparation-adipose-progenitor-cells-from-mouse-epididymal-adipose

Nanyang Technological University

Research

JoVE Journal

Biology

5.5K 次观看.  Mayo Clinic. 我们提出了一个简单的方法，利用荧光激活细胞分拣从小鼠表皮脂肪垫中分离出高度可行的青春期祖细胞。

视频: Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

科研

生物学

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

人主动脉周脂肪祖细胞的分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

各种鼠标脂肪库的识别和解剖

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

通过甲基异硫清除和三D成像探索脂肪组织结构

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

恒河猴脂肪干细胞的分离、增殖和分化

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

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

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

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

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

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 ...

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 ...