Name: preparation of adipose progenitor cells from mouse epididymal adipose tissues
Uploaded: 2020-08-25T14:45:00
Description: Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

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 ?...

<ol>
	<li>Krotkiewski, M., Bjorntorp, P., Sjostrom, L., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+obesity+on+metabolism+in+men+and+women.+Importance+of+regional+adipose+tissue+distribution.">Impact of obesity on metabolism in men and women. Importance of regional adipose tissue distribution.</a> The Journal of Clinical Investigation. 72, 1150-1162 (1983).</li><li>Salans, L. B., Horton, E. S., Sims, E. 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P., Scheller, E. L., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+stem+cells+meet+preadipocyte+commitment:+going+back+to+the+future.">Adipose tissue stem cells meet preadipocyte commitment: going back to the future.</a> Journal of Lipid Research. 53, 227-246 (2012).</li><li>Cho, D. S., Lee, B., Doles, 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=Refining+the+adipose+progenitor+cell+landscape+in+healthy+and+obese+visceral+adipose+tissue+using+single-cell+gene+expression+profiling.">Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.</a> Life Science Alliance. 2, 201900561 (2019).</li><li>Burl, R. B., 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=Deconstructing+Adipogenesis+Induced+by+beta3-Adrenergic+Receptor+Activation+with+Single-Cell+Expression+Profiling.">Deconstructing Adipogenesis Induced by beta3-Adrenergic Receptor Activation with Single-Cell Expression Profiling.</a> Cell Metabolism. 28, 300-309 (2018).</li><li>Hepler, C., 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=Identification+of+functionally+distinct+fibro-inflammatory+and+adipogenic+stromal+subpopulations+in+visceral+adipose+tissue+of+adult+mice.">Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.</a> eLife. 7, 39636 (2018).</li><li>Merrick, D., 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=Identification+of+a+mesenchymal+progenitor+cell+hierarchy+in+adipose+tissue.">Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.</a> Science. 364, 2501 (2019).</li><li>Schwalie, P. C., 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=A+stromal+cell+population+that+inhibits+adipogenesis+in+mammalian+fat+depots.">A stromal cell population that inhibits adipogenesis in mammalian fat depots.</a> Nature. 559, 103-108 (2018).</li><li>Raajendiran, 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=Identification+of+Metabolically+Distinct+Adipocyte+Progenitor+Cells+in+Human+Adipose+Tissues.">Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues.</a> Cell Reports. 27, 1528-1540 (2019).</li><li>De Matteis, R., 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=In+vivo+physiological+transdifferentiation+of+adult+adipose+cells.">In vivo physiological transdifferentiation of adult adipose cells.</a> Stem Cells. 27, 2761-2768 (2009).</li><li>Hanamsagar, R., 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=An+optimized+workflow+for+single-cell+transcriptomics+and+repertoire+profiling+of+purified+lymphocytes+from+clinical+samples.">An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples.</a> Scientific Reports. 10, 2219 (2020).</li><li>Marinovic, M. 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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?...

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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	<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...

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

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从人类Lipoaspirates脂肪来源干细胞的手动隔离

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人主动脉周脂肪祖细胞的分化能力

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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

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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) ...

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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 ...

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