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?...

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><tbody><tr><td>1.7 mL microcentrifuge tube</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>13 mL culture tube</td><td>Thermo Fisher Scientific</td><td>50-809-216</td><td></td></tr><tr><td>15 mL conical tube</td><td>Greiner Bio-one</td><td>188 271</td><td></td></tr><tr><td>5 mL test tube with cell strainer snap cap</td><td>Thermo Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner Bio-one</td><td>227 261</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Thermo Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>Anti-CD31-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-519</td><td></td></tr><tr><td>Anti-CD45-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-491</td><td></td></tr><tr><td>Anti-Sca1-APC antibody</td><td>Miltenyi Biotec</td><td>130-102-833</td><td></td></tr><tr><td>Anti-Ter119-FITC antibody</td><td>Miltenyi Biotec</td><td>130-112-908</td><td></td></tr><tr><td>BSA</td><td>Gold Biotechnology</td><td>A-420-500</td><td></td></tr><tr><td>Collagenase type II</td><td>Thermo Fisher Scientific</td><td>17101-015</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190-144</td><td></td></tr><tr><td>F-12 medium</td><td>Thermo Fisher Scientific</td><td>11765-054</td><td></td></tr><tr><td>FcR blocking reagent</td><td>Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>Hanks&#039; balanced salt solution (HBSS)</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050-122</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Propidium iodide solution</td><td>Miltenyi Biotec</td><td>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...

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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 ...

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5.6K 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 Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

科研

发育生物学

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. ...

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

<meta charset="utf8"></head><body>分离和加工小鼠白脂肪细胞用于转录组和表观基因组分析

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

分离和加工小鼠白脂肪细胞用于转录组和表观基因组分析

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

遗传学

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 ...

免疫与感染

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

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

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 and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

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

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

初白和棕色预生细胞与新生小鼠的隔离和分化

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

生物学

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

原代培养中分化的小鼠脂肪细胞:胰岛素抵抗模型

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

培养从小鼠血管周围脂肪组织中分离的前脂肪细胞

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

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

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

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

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通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

隔离和脂肪干细胞分化的猪从皮下脂肪组织

分离和加工小鼠白脂肪细胞用于转录组和表观基因组分析

分离和加工小鼠白脂肪细胞用于转录组和表观基因组分析

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

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

初白和棕色预生细胞与新生小鼠的隔离和分化

原代培养中分化的小鼠脂肪细胞:胰岛素抵抗模型

从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞

从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞