脂肪库专用SCA1的免疫磁性分离<sup>高</sup>脂肪干细胞（ASCs）
Summary
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.
Abstract
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.
Introduction
干细胞抗原-1（SCA1，或Ly6A / E）最初被鉴定为通过造血细胞和间质干细胞5,6-表达的细胞表面标记。从小鼠脂肪库获得脂肪组织的基质血管级分（SVF）为含有成纤维细胞，巨噬细胞，血管内皮细胞，神经细胞，以及脂肪细胞祖细胞7的细胞的异质群体。脂肪细胞祖细胞，或脂肪干细胞（ASCs）是驻留在富含胶原蛋白的血管周围的细胞外基质（ECM）8非载脂细胞。大约SVF的50％由携带者，其特点为谱系阴性（林– ）和CD29 +：CD34 +：SCA1 + 9。大多数这些细胞是SCA1 +：CD24 –脂肪细胞祖细胞，其能够在体外脂肪细胞分化的;然而，只有细胞的级分（SVF的0.08％）构成SCA1 <sUP> +：CD24 +细胞完全有能力和增殖分化为体内条件下9脂肪细胞。尽管使用SCA1 + SVF不脱离CD24识别CD24 +细胞的电位警告–的细胞，分离SCA1 +的ASC从用免疫细胞分离脂肪库是一种高效，实用的方法，以确定主脂肪细胞祖细胞的细胞自主表型。
在肥胖和糖尿病，组织纤维化和炎症的领域发挥在2型糖尿病3的开发和维护的关键作用。最近，德永等人 。表明，腹股沟（或皮下，SQ）和perigonadal（或内脏，VIS）隔离SCA1 高细胞C57BL6 / J脂肪库表现出体外 10个不同的基因签名和ECM重塑。 MMP14（MT1-MMP），膜 – t的典型构件YPE基质金属蛋白酶（MMP）家族通过其原活性1介导的白色脂肪组织（WAT）的开发。
可与分离和富集通过以下方案中的细胞进行的实验的例子包括三维培养，分化研究，胶原降解测定法，和RNA测序10,11。降解测定法应与酸提取胶原进行，以确保端肽11,12的保存。下面的协议将演示方法主要血管基质细胞从不同的脂肪库分离和使用免疫细胞分离脂肪细胞丰富的祖细胞。在细胞分选的有效性将用流式细胞仪，并通过使用SCA1-GFP小鼠表达SCA1 +细胞GFP，由SCA1启动子驱动13进行评估。
Protocol
Representative Results
Discussion
在此，我们展示了隔离，并从不同的脂肪垫鼠携带者的免疫细胞分离和体外实验。所提出的方法是有效的的大量SCA1阳性的ASC的，这是过度的ASC 9,14的技术复杂和昂贵的FACS介导的隔离有利的快速隔离。不像FACS，免疫细胞分离不允许靶细胞群体的鉴定使用多个抗原。然而，如果表面抗原表征良好，利用免疫磁性分离的增加不依赖于使用的FACS设备15，其仍然不小院所许多生物学?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院DK095137（THC到）的支持。我们感谢现任和前任实验室成员是谁所描述的方法的发展和成熟作出了贡献。
Materials
| Type 3 Collagenase | Worthington Biochemical | LS004182 | Tissue digestion |
| DMEM | Gibco | 11965-092 | High-glucose culture medium |
| Pen/Strep/Glutamine (100x) | Gibco | 10378-016 | Media antibiotic |
| Anti-anti (100x) | Gibco | 15240-062 | Media antifungal |
| FBS | Gibco | 16000-044 | |
| PBS (1x, pH 7.4) | Gibco | 10010-023 | |
| Trypsin (0.05%) | Gibco | 25300-054 | |
| Cell strainer | BD Bioscience | 352360 | 100-μm cell strainer |
| 60mm plates | BD Falcon | 353004 | |
| Scissors | FST | 14001-12 | Large |
| Scissors | FST | 14091-11 | Fine, curved tip |
| Large Forceps | FST | 11000-12 | |
| Fine Forceps | Any vendor | ||
| 25G 5/8” needles | BD | 305122 | |
| 22G 1.5” needles | BD | 305159 | |
| 15 ml conical tubes | BD Falcon | 352097 | |
| 50 ml conical tubes | BD Falcon | 352098 | |
| MACS separation columns | Miltenyi Biotec | 130-042-201 | |
| Anti-Sca1 microbead kit (FITC) | Miltenyi Biotec | 130-092-529 | FITC-anti-Sca1 1ºAb and anti-FITC microbeads 2ºAb |
| AutoMACS running buffer | Miltenyi Biotec | 130-091-221 | |
| MiniMACS separator | Miltenyi Biotec | 130-042-102 | |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
| Blue chux pads | Fisher | 276-12424 | |
| Absorbent pads | Fisher | 19-165-621 | |
| Styrofoam board | Use from 50ml tubes | ||
| 70% ethanol | |||
| Isoflurane | Any vendor | ||
| Rat IgG2a Alexa Fluor 647 | Invitrogen | R2a21 | |
| Rat IgG2a anti-mouse Sca1 Alexa Fluor 647 | Invitrogen | MSCA21 | |
| Rat IgG2a R-PE | Invitrogen | R2a04 | |
| Rat IgG2a anti-mouse F4/80 R-PE | Invitrogen | MF48004 | |
| Round-bottom tube | BD Falcon | 352058 | |
| HBSS (–Ca, –Mg) | Gibco | 14175-095 | |
| HBSS (+Ca, +Mg) | Gibco | 14025-092 | For collagenase solution |
| Type I collagen (2.7 mg/ml in 37mm acetic acid | Prepare in house12 | ||
| 10x MEM | Gibco | 11430-030 | |
| 1M HEPES | Gibco | 15630-080 | |
| 0.34N NaOH | Prepare in house | ||
| Cover slips | Corning | 2870-22 | |
| Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers | Invitrogen | A-20004 | |
| 0.89M NaHCO3 | Gibco | 25080-094 |
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