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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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+study+of+adipocyte+precursors.">Isolation and study of adipocyte precursors.</a> Methods Enzymol. 537, 31-46 (2014).</li><li>Buffolo, M., 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+Paracrine+Signaling+Mechanism+Linking+CD34(high)+Progenitors+to+the+Regulation+of+Visceral+Fat+Expansion+and+Remodeling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+adipocyte+differentiation+from+Pdgfrbeta(+)+preadipocytes+protects+against+pathologic+visceral+adipose+expansion+in+obesity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly6+family+proteins+in+neutrophil+biology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+human+adipose+tissue+identifies+depot+and+disease+specific+cell+types.">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>Mechanical Tissue Preparation and SVF Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 and 100 &#181;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>Digestion Buffer (for 10mL)</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>Immunomagnetic separation of APCs and non-APCs</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 &#181;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>Purity Check and FACS</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>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin CD45</td>
			<td>BioLegend</td>
			<td>103103</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>Biotin CD31</td>
			<td>BioLegend</td>
			<td>102503</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MEC13.3</td>
		</tr>
		<tr>
			<td>Biotin CD9</td>
			<td>BioLegend</td>
			<td>124803</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td>Biotin LY6C</td>
			<td>BioLegend</td>
			<td>128003</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD31-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>102419</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 390</td>
		</tr>
		<tr>
			<td>CD45-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>103131</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>CD140b PDGFR&#946;-PE</td>
			<td>BioLegend</td>
			<td>136006</td>
			<td>Concentration: Dilution 1:50 
			Species: Mouse 
			Clone: APB5</td>
		</tr>
		<tr>
			<td>LY6C-APC</td>
			<td>BioLegend</td>
			<td>128016</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD9-FITC</td>
			<td>BioLegend</td>
			<td>124808</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture and Differentiation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gonadal APC Culture media (for 500mL)</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 &#181;L 100 g/ml FGF-basic</td>
			<td>R&#38;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 &#181;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

2.4K 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 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 ...

科研

免疫与感染

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

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 ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

从人类和小鼠心肌标本中分离巨噬细胞子集和细胞位细胞

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol&#160;reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

从小鼠肩胛间棕色脂肪组织中分离棕色脂肪细胞以进行基因和蛋白质表达分析

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs ...