作者感谢 Lisa Hansen 和 Kirsten Vestergaard 提供的出色技术援助，以及 P. Scherer、N. Joffin 和 C. Crewe 对手稿的批判性阅读。作者感谢UTSW流式细胞术核心在制定本文所述方案方面提供的出色指导和帮助。R.K.G. 受 NIH NIDDK R01 DK104789、NIDDK RC2 DK118620 和 NIDDK R01 DK119163 支持。J.P.由丹麦创新基金的博士前奖赞助。

所有动物协议和程序均已获得德克萨斯大学西南医学中心机构动物使用和护理委员会的批准。
1. 从性腺白色脂肪组织中分离基质血管分数 （SVF）
<ol><li>解剖来自6-8周龄小鼠的性腺白色脂肪组织，并将脂肪垫置于1x PBS溶液中。</li>
	<li>结合多达 4 个脂肪库（推荐 1-2 只小鼠的 2-4 个脂肪库），并在含有 200 μL 消化缓冲液（1x HBSS，1.5% BSA 和 1 mg/mL 胶原酶 D）的 10 mL 烧杯中切碎组织。</li>
	<li>将切碎的组织转移到含有 10 mL 消化缓冲液的 50 mL 离心管中。</li>
	<li>在37°C的水浴中孵育混合物1小时，同时摇动。</li>
	<li>通过100μm细胞过滤器过滤消化组织以去除未消化的组织。</li>
	<li>用含有2%FBS的PBS将过滤样品稀释至30mL，并在4°C下以600 ×g 离心5分钟。 吸出含有成熟脂肪细胞的上清液。</li>
	<li>立即进行首选的细胞分离方法。</li>
</ol>2. 使用FACS分离APC和FIP
<ol><li>轻轻摇动试管，将沉淀溶解在 1 mL 1x RBC 裂解缓冲液中。</li>
	<li>在室温下孵育 1-2 分钟。</li>
	<li>加入 10 mL 2% FBS/PBS，并通过 40 μm 细胞过滤器进入干净的 50 mL 离心管中。</li>
	<li>在4°C下以600 ×g 离心5分钟。</li>
	<li>吸出培养基并将沉淀重悬于含有 Fc 块 （1:200） 的 400-800 μL 2% FBS/PBS 中。</li>
	<li>在4°C孵育10分钟。</li>
	<li>将 400 μL-800 μL 含有 ≤106 个细胞/mL 的细胞悬液转移到 1.5 mL 试管中，并加入抗体。抗体浓度列在 "材料表 "部分。<ol><li>为 1） 未染色的细胞、2） 单色对照和 3） FMO（荧光减一）对照准备单独的对照管。</li>
		<li>用PDGFRβ，CD45，CD31，CD9，LY6C抗体对样品进行染色。</li>
	</ol></li>
	<li>在4°C下避光孵育15分钟。</li>
	<li>在4°C下以600 ×g 离心5分钟。</li>
	<li>吸出培养基并将细胞重悬于 400 μL 2% FBS/PBS 中。</li>
	<li>在4°C下以600 ×g 离心5分钟。</li>
	<li>吸出培养基并将细胞重悬于 400-800 μL 2% FBS/PBS 中。然后将 40 μm 滤帽放入 5 mL 聚苯乙烯圆底管中用于 FACS。</li>
	<li>按照以下步骤进行门控。<ol><li>使用无染色和单色对照进行补偿。</li>
		<li>使用 FMO 控件设置实验门。</li>
		<li>使用图 1 中所示的门控策略获取 APC 和 FIP。门 APC 为 CD45-CD31-PDGFRβ+ LY6C-CD9- 和 FIPs 为 CD45-CD31-PDGFRβ+ LY6C+ 细胞。</li>
	</ol></li>
	<li>将分选的细胞收集在含有 500 μL 100% 血清的试管中，并将细胞保持在冰上。</li>
	<li>在4°C下以600 ×g 离心细胞5分钟。 加入2%FBS / PBS通过在4°C下再次以600 ×g 离心5分钟来洗涤细胞。 丢弃上清液，使用沉淀的细胞。</li>
	<li>将沉淀的细胞重悬于适当体积的性腺APC培养基（用于细胞培养）或适当的缓冲液中，以便进行后续分析。</li>
</ol>3. 成脂和非成脂部分的免疫磁性分离
<ol><li>按照步骤 1.1-1.7 从性腺白色脂肪组织中分离 SVF。</li>
	<li>吸出上清液并将 SVF 沉淀重悬于 10 mL 1x 市售 MS 缓冲液中。</li>
	<li>通过 40 μm 细胞过滤器过滤细胞悬浮液。</li>
	<li>在4°C下以600 ×g 离心5分钟。</li>
	<li>吸出上清液并将沉淀的细胞重悬于 100 μL 的 1x MS 缓冲液中。</li>
	<li>如下所述进行CD31 + 和CD45 + 细胞耗竭（图2A， 步骤1）。这样做是为了去除内皮细胞和造血谱系细胞。<ol><li>使用细胞计数器计数细胞，并将浓度调节至 ≤ 1 x 108 个细胞/mL。</li>
		<li>将生物素偶联的 CD31 和 CD45 抗体加入细胞悬液中，每个抗体的浓度为每 106 个细胞 ≤ 0.25 μg，并在冰上孵育 15 分钟。</li>
		<li>通过加入 4 mL 1x MS 缓冲液洗涤细胞。</li>
		<li>在4°C下以600 ×g 离心5分钟。</li>
		<li>吸出上清液并用 100 μL 1x MS 缓冲液重悬沉淀。</li>
		<li>加入 10 μL 链霉亲和素纳米珠，将细胞在冰上孵育 15 分钟。</li>
		<li>通过加入 4 mL 1x MS 缓冲液洗涤细胞。</li>
		<li>在4°C下以600 ×g 离心5分钟，然后吸出上清液。</li>
		<li>将细胞重悬于 2.5 mL 1x MS 缓冲液中，并转移到 5 mL 聚丙烯管中。</li>
		<li>将管子放入磁铁架中 5 分钟。</li>
		<li>通过将细胞悬浮液倒入干净的 15 mL 离心管中来收集未标记的细胞。 注意:避免摇晃或吸干悬挂的液滴，因为这会导致不需要的细胞部分污染。</li>
		<li>从磁铁上取下管子并重复步骤 3.6.9。到 3.6.11。再洗两次，共洗 3 次。</li>
	</ol></li>
	<li>脂肪生成和非脂肪生成部分的分离（图2A， 步骤2）<ol><li>继续处理未标记的馏分（即CD45-CD31-馏分）。</li>
		<li>将含有未标记细胞的15mL管在4°C下以600 ×g 离心5分钟。</li>
		<li>吸出上清液并将沉淀重悬于 100 μL 1x MS 缓冲液中。</li>
		<li>加入生物素偶联的LY6C和CD9抗体，浓度分别为每106 个细胞≤0.25μg，并在冰上孵育15分钟。</li>
		<li>按照步骤 3.6.3 操作。到 3.6.12。完成第二次分离。</li>
		<li>收集未结合的分数。未标记的细胞（LY6C-CD9-）代表含有APC的脂肪生成部分（图2A，步骤3）。</li>
		<li>从磁体中取出试管，重悬并在 1x MS 缓冲液中洗脱结合的细胞。洗脱液或标记的馏分（LY6C + CD9 +）代表含有FIPs的非成脂级分（图2A， 步骤3）。</li>
		<li>将标记和未标记的馏分以600 ×g 离心5分钟，以在4°C下沉淀细胞。</li>
	</ol></li>
	<li>立即进行细胞培养（第 6 步）或使用未分化的前体进行首选分析（第 4 步和/或第 5 步）。</li>
</ol>4. 通过流式细胞术评估成脂和非成脂部分的纯度
<ol><li>将珠子分离的细胞级分重悬于 200-400 μL 含有 Fc 块 （1:200） 的 2% FBS/PBS 中。</li>
	<li>在4°C孵育10分钟。</li>
	<li>将细胞悬液转移到 1.5 mL 试管中并添加抗体。抗体浓度列在 材料表中。<ol><li>为 1） 未染色细胞、2） 单色对照和 3） FMO 对照准备单独的对照管。</li>
		<li>向样品中加入 CD45、CD31、CD9 和 LY6C 抗体。</li>
	</ol></li>
	<li>在4°C下避光孵育15分钟。</li>
	<li>在4°C下以600 ×g 离心5分钟。</li>
	<li>吸出上清液并将细胞重悬于 400 μL 2% FBS/PBS 中。</li>
	<li>将悬浮液在4°C下以600 ×g 离心5分钟。</li>
	<li>吸出上清液并将细胞重悬于 400-800 μL 2% FBS/PBS 中。通过 40 μm 滤帽过滤到 5 mL 聚苯乙烯圆底管中，通过流式细胞术进行分析。</li>
	<li>流式细胞术分析以评估纯度<ol><li>使用无染色和单色对照进行补偿。</li>
		<li>使用 FMO 控件设置实验门。</li>
		<li>对活细胞和单细胞使用门控策略， 如图2B所示。</li>
		<li>如图 2B 所示对 CD45、CD31、LY6C 和 CD9 执行门控。</li>
		<li>使用流式细胞术软件评估分离组分中 CD45-CD31-LY6C-CD9- 细胞 （APC） 和 CD45-CD31-LY6C+ 细胞 （FIP） 的频率。</li>
	</ol></li>
</ol>5. 使用定量 PCR 进行基因表达分析，以评估 FIP 和 APC 的纯度
<ol><li>按照制造商的说明，使用市售的提取试剂盒（参见 材料表）从磁性或 FACS 分离的细胞中提取 mRNA。</li>
	<li>按照制造商的说明，使用 1 μg RNA 使用 cDNA 逆转录酶试剂盒（参见 材料表）合成 cDNA。</li>
	<li>使用SYBR绿色PCR预混液通过定量PCR确定APC和FIPs选择性基因的相对mRNA水平（表1）。<ol><li>设置用于PCR的样品反应如下:5μL的2x SYBR绿色染料，1μL的cDNA（~50ng/μL），0.5μL的每种正向和反向引物（10μM）和3μL的水。</li>
		<li>使用以下标准PCR条件进行定量PCR运行:保持阶段:50°C2分钟，95°C10分钟;PCR阶段（40个周期）:95°C15秒，60°C1分钟。</li>
	</ol></li>
	<li>通过计算 ΔΔ-Ct 将值归一化为管家基因 （Rps18） 水平。</li>
	<li>要评估统计显著性，请执行未配对的学生 t 检验。</li>
</ol>6. 细胞培养和分化
<ol><li>在4°C下以600 ×g 离心磁性或FACS-分离的细胞5分钟。</li>
	<li>将沉淀重悬于 500 μL 性腺 APC 培养基中，并在 48 孔培养板中将每个级分的 40K 个细胞/孔铺板。</li>
	<li>每 1-2 天更换一次培养基。当 APC 接近汇合时，它们将开始分化为脂肪细胞。在相同培养基中维持的 FIP 对脂肪生成具有抵抗力。</li>
</ol>

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<li>Buffolo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+Paracrine+Signaling+Mechanism+Linking+CD34%28high%29+Progenitors+to+the+Regulation+of+Visceral+Fat+Expansion+and+Remodeling%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Identification of a Paracrine Signaling Mechanism Linking CD34(high) Progenitors to the Regulation of Visceral Fat Expansion and Remodeling.</a> Cell Reports. 29 (2), 270-282 (2019).</li>
<li>Shao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+adipocyte+differentiation+from+Pdgfrbeta%28%2B%29+preadipocytes+protects+against+pathologic+visceral+adipose+expansion+in+obesity%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">De novo adipocyte differentiation from Pdgfrbeta(+) preadipocytes protects against pathologic visceral adipose expansion in obesity.</a> Nature Communications. 9 (1), 890(2018).</li>
<li>Lee, P. Y., Wang, J. X., Parisini, E., Dascher, C. C., Nigrovic, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ly6+family+proteins+in+neutrophil+biology%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">Ly6 family proteins in neutrophil biology.</a> Journal of Leukocyte Biology. 94 (4), 585-594 (2013).</li>
<li>Vijay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+analysis+of+human+adipose+tissue+identifies+depot+and+disease+specific+cell+types%5BTitle%5D)+AND+%22Nature+Metabolism%22%5BJournal%5D)">Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.</a> Nature Metabolism. 2 (1), 97-109 (2020).</li>
</ol>

T.A.P. 是 Novo Nordisk A/S 的员工和股东

小鼠C57BL / 6品系是饮食诱导的肥胖研究中使用最多的小鼠品系。C57BL/6小鼠在高脂肪饮食（HFD）时体重迅速增加，并出现与肥胖相关的代谢综合征的一些突出特征（例如，胰岛素抵抗和高脂血症）。值得注意的是，与高脂肪饮食 （HFD） 喂养相关的 WAT 扩增以仓库特异性方式发生 19,20,21,22,23。<su...

白色脂肪组织（WAT）是哺乳动物储存能量的主要场所。在这种组织中，脂肪细胞或"脂肪细胞"以甘油三酯的形式储存多余的卡路里，这些卡路里被包装成大的单房脂滴。此外，脂肪细胞分泌多种因子来调节能量稳态的各个方面 1,2,3。脂肪细胞占WAT体积的大部分;然而，脂肪细胞仅占 WAT 4,5 中发现的总细胞的不到 50%。WAT 的非脂肪细胞区室或基质血管部分 （SVF） 具有相当异质性，包含血管内皮细胞、组织驻留免疫细胞、成纤维细胞和脂肪细胞前体细胞 （APC） 群体。
随着对储能需求的增加，WAT具有出色的扩展能力。保持这种组织可塑性至关重要，因为在 WAT 中充分储存脂质可以防止有害的异位脂质沉积到非脂肪组织中6.在肥胖的情况下，各个 WAT 仓库因热量过剩而进行这种扩张的方式是胰岛素敏感性的关键决定因素7.在患有代谢综合征的肥胖个体中观察到的病理性 WAT 扩张，其特征是内脏 WAT 库优先扩张，而牺牲代谢有利的皮下脂肪组织。此外，肥胖症中的胰岛素抵抗与 WAT 的病理重塑有关。其特征是现有脂肪细胞的肥大生长（尺寸增加），血管生成不足，慢性代谢炎症，细胞外基质成分（纤维化）的积累和组织缺氧8,9。这些肥胖的 WAT 表型与肝脂肪变性和胰岛素抵抗有关，类似于在脂肪营养不良（缺乏功能性 WAT）条件下观察到的情况。相比之下，在代谢健康的肥胖人群中观察到健康的 WAT 扩张，其特征是保护性皮下 WAT 的优先扩张和通过脂肪细胞增生的仓库扩张10。新脂肪细胞的募集是通过从脂肪细胞前体细胞 （APC） 中新头分化脂肪细胞介导的（称为"脂肪生成"）。脂肪细胞增生与相对较低程度的 WAT 纤维化和代谢炎症相吻合 6,11。WAT 微环境中的多种细胞类型直接影响 WAT 在肥胖中的健康和可扩展性12.因此，定义WAT中存在的各种细胞类型的功能仍然是该领域的重中之重。
在过去的十年中，已经采用了几种策略来定义和分离来自人和小鼠 WAT SVF13 的天然 APC。这些策略使用基于抗体的细胞分离技术，根据常见间充质干细胞/祖细胞标志物的细胞表面表达分离 APC。这些方法包括使用荧光团标记抗体的荧光激活细胞分选 （FACS） 或免疫磁珠分离（即化学修饰的抗体）。靶向分离 APC 的细胞表面蛋白包括 PDGFRα、PDGFRβ、CD34 和 SCA-1。这些方法有助于丰富APC;然而，基于这些标记物分离的细胞群具有相当大的异质性。最近的单细胞 RNA 测序 （scRNA-seq） 研究强调了小鼠 WAT 14,15,16,17 分离的基质-血管部分 （SVF） 内基质细胞的分子和功能异质性。根据我们自己的 scRNA-seq 和功能分析，我们已经鉴定并表征了成年小鼠腹内 WAT 基质区室中功能不同的免疫调节和成脂 PDGFRβ+ 血管周围细胞亚群15。纤维炎症前体 （FIP） 是 PDGFRβ+ 细胞的重要亚群，可以根据 LY6C 表达（LY6C+ PDGFRβ+ 细胞）15 进行分离。FIPs缺乏成脂能力，对各种刺激产生强烈的促炎反应，产生胶原蛋白，并分泌抗脂肪生成因子15。这些细胞的促炎和纤维化活性与小鼠肥胖有关而增加，表明这些细胞是WAT重塑的调节因子。LY6C-CD9-PDGFRβ+ 亚群代表脂肪细胞前体细胞 （APC）。这些 APC 富含 Pparg 和其他促脂肪基因的表达，并且在体外和体内很容易分化成成熟的脂肪细胞15。在这里，我们提供了一个详细的方案，用于使用FACS从成年小鼠的腹内WAT库中分离这些不同的细胞群，并使用生物素化抗体进行免疫磁珠分离。该协议可用于从成年雄性和雌性小鼠的多个腹内WAT库中分离功能不同的脂肪祖细胞亚群15。单独研究这些功能不同的细胞群可能极大地有助于我们目前对健康和疾病中调节脂肪生成和腹内脂肪组织重塑的分子机制的理解。
以下方案详细介绍了从小鼠附睾WAT中分离脂肪祖细胞的方法;然而，相同的程序可用于从雄性和雌性小鼠的肠系膜和腹膜后 WAT 库中分离相应的细胞15。关于如何在小鼠中识别和分离这些仓库的详细方案可以在Bagchi等人18中找到。该协议已针对6-8周龄的小鼠的使用进行了优化。APCs的频率和分化能力可能随着年龄的增长而下降。

<table><tbody><tr><td>&lt;strong&gt;Mechanical Tissue Preparation and SVF Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>40 and 100 &amp;micro;m cell strainers</td><td>Fisher Scientific</td><td>352340/352360</td><td></td></tr><tr><td>1X Phosphate buffered saline (PBS)</td><td>Fisher Scientific</td><td>21040CV</td><td></td></tr><tr><td>5ml polypropylene tubes</td><td>Fisher Scientific</td><td>352053</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Digestion Buffer (for 10mL)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 ml HBSS</td><td>Sigma</td><td>H8264</td><td></td></tr><tr><td>10 mg Collagenase D (1 mg/ml final cc.)</td><td>Roche</td><td>11088882001</td><td></td></tr><tr><td>0.15 g BSA (1.5 % final cc.)</td><td>Fisher Scientific</td><td>BP1605-100</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Immunomagnetic separation of APCs and non-APCs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5X MojoSort Buffer (MS buffer)</td><td>BioLegend</td><td>480017</td><td></td></tr><tr><td>5 ml MojoSort Magnet (MS magnet)</td><td>BioLegend</td><td>480019</td><td></td></tr><tr><td>100 &amp;micro;L MojoSort Streptavidin Nanobeads</td><td>BioLegend</td><td>480015</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Purity Check and FACS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X Red Blood Cell Lysis Buffer</td><td>eBioscience</td><td>00-4300-54</td><td></td></tr><tr><td>Fc block (Mouse CD16/CD32)</td><td>eBioscience</td><td>553141</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biotin CD45</td><td>BioLegend</td><td>103103</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: 30-F11</td></tr><tr><td>Biotin CD31</td><td>BioLegend</td><td>102503</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: MEC13.3</td></tr><tr><td>Biotin CD9</td><td>BioLegend</td><td>124803</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: MZ3</td></tr><tr><td>Biotin LY6C</td><td>BioLegend</td><td>128003</td><td>Concentration: &amp;le; 0.25 &amp;micro;g per 10^6 cells&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: HK1.4</td></tr><tr><td>CD31-PerCP/Cy5.5</td><td>BioLegend</td><td>102419</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: 390</td></tr><tr><td>CD45-PerCP/Cy5.5</td><td>BioLegend</td><td>103131</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: 30-F11</td></tr><tr><td>CD140b PDGFR&amp;beta;-PE</td><td>BioLegend</td><td>136006</td><td>Concentration: Dilution 1:50&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: APB5</td></tr><tr><td>LY6C-APC</td><td>BioLegend</td><td>128016</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: HK1.4</td></tr><tr><td>CD9-FITC</td><td>BioLegend</td><td>124808</td><td>Concentration: Dilution 1:400&lt;br/&gt; Species: Mouse&lt;br/&gt; Clone: MZ3</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell Culture and Differentiation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Gonadal APC Culture media (for 500mL)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>288 mL DMEM with 1 g/L glucose</td><td>Corning</td><td>10-014-CV</td><td></td></tr><tr><td>192 mL MCDB201</td><td>Sigma</td><td>M6770</td><td></td></tr><tr><td>10 mL Fetal bovine serum (FBS)** lot#14E024</td><td>Sigma</td><td>12303C</td><td></td></tr><tr><td>5 mL 100% ITS premix</td><td>BD Bioscience</td><td>354352</td><td></td></tr><tr><td>5 mL 10 mM L-ascorbic acid-2-2phosphate</td><td>Sigma</td><td>A8960-5G</td><td></td></tr><tr><td>50 &amp;micro;L 100 g/ml FGF-basic</td><td>R&amp;amp;D systems</td><td>3139-FB-025/CF</td><td></td></tr><tr><td>5 mL Pen/Strep</td><td>Corning</td><td>30-001-CI</td><td></td></tr><tr><td>500 &amp;micro;L Gentamycin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>**NOTE: The adipogenic capacity of primary APCs can vary from lot to lot of commercial FBS. Multiple lots/sources of FBS should be tested.</td><td></td><td></td><td></td></tr></tbody></table>

isolation of adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal adipose depots

白色脂肪组织 （WAT） 的基质-血管部分 （SVF） 具有显着的异质性，由多种细胞类型组成，这些细胞类型在功能上有助于成年期 WAT 的扩增和重塑。研究这种细胞异质性的影响的一个巨大障碍是无法轻易地从 WAT SVF 中分离出功能不同的细胞亚群以进行体外和体内分析。单细胞测序技术最近在成年小鼠腹内WAT库中鉴定出功能不同的纤维炎和成脂PDGFRβ+血管周围细胞亚群。纤维炎祖细胞（称为"FIP"）是非脂肪生成胶原蛋白产生细胞，可以发挥促炎表型。PDGFRβ+ 脂肪细胞前体细胞 （APC） 在细胞移植后在体外和体内都具有高度脂肪生成性。在这里，我们描述了从小鼠腹内 WAT 仓库中分离这些基质细胞亚群的多种方法。FIP 和 APC 可以通过荧光激活细胞分选 （FACS） 或利用基于生物素化抗体的免疫磁珠技术进行分离。分离的细胞可用于分子和功能分析。孤立地研究基质细胞亚群的功能特性将扩展我们目前在细胞水平的生理或病理条件下脂肪组织重塑的知识。

该协议描述了两种策略，允许从成年小鼠的腹内WAT仓库中分离不同的基质细胞群。APC和FIP可以通过FACS（图1）或使用生物素化抗体（图2）分离免疫磁珠来分离。这两种方法都利用了市售的试剂和抗体。免疫磁珠分离可从 gWAT SVF 中分离出成脂细胞和非成脂细胞。流式细胞术分析显示，成脂部分内75%的细胞代表LY6C-CD9-APCs。>75% 的非成脂部?...

Read this Scientific Article Protocol about Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots at JoVE.com

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

该协议描述了通过荧光激活细胞分选或免疫磁珠分离从小鼠腹内白色脂肪组织（WAT）库中分离脂肪生成和纤维炎基质细胞亚群的技术方法。

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

isolation-adipogenic-fibro-inflammatory-stromal-cell-subpopulations

Research

JoVE Journal

Immunology and Infection

2.7K 次观看.  Novo Nordisk A/S. 该协议描述了通过荧光激活细胞分选或免疫磁珠分离从小鼠腹内白色脂肪组织（WAT）库中分离脂肪生成和纤维炎基质细胞亚群的技术方法。

视频: Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

科研

发育生物学

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

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 ...

免疫与感染

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

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

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 ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

医学

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

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 ...

生物学

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 ...

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

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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

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

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