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Isolation of LYVE-1+ Endothelial Cells from Mouse Embryos

从小鼠胚胎中分离LYVE-1+内皮细胞的方法

PC Patrick Crosswhite

发布: 2018年08月05日第8卷第15期 DOI: 10.21769/BioProtoc.2962 浏览次数: 6159

评审: Jia LiJijun ChengAnonymous reviewer(s)

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Abstract

Lymphatic vessel endothelial hyaluronan receptor 1, or LYVE-1, is a type 1 integral membrane glycoprotein expressed by lymphatic endothelial cells (LECs). LYVE-1 is commonly used as a biological marker to visually distinguish developing lymphatic vessels from blood endothelial cells (arteries or veins). As our understanding of lymphatic biology is still lacking today, the need to isolate LECs apart from other endothelial cells has taken on greater importance. The following procedure describes a magnetic bead separation procedure for isolating LEC-rich populations of cells from developing mouse embryos.

Keywords: LYVE-1 (LYVE-1) search Endothelial cells (内皮细胞) search Lymphatic (淋巴) search Liver (肝) search Mouse embryo (小鼠胚胎) search

Background

The lymphatic vasculature forms a secondary circulatory system that functions in draining extracellular fluid from tissue, allows for the transport of lipids, and provides immune cell trafficking and transport function. While our understanding of the lymphatic system has rapidly expanded in the last couple of decades, it is still lacking compared to our knowledge of arterial and venous biology. The identification of LYVE-1 receptor expression by LECs provided a useful tool to distinguish lymphatic tissue but LYVE-1 is known to be expressed in other tissues including liver and spleen sinusoidal cells and pancreatic exocrine and islet of Langerhans cells (Banerji et al., 1999). Encoded by the LYVE1 gene, the biological function of the LYVE-1 receptor has yet to be determined but it has been suggested to participate in tumor metastasis in addition to HA transport across endothelial cells (Jackson, 2003). Despite it being expressed in a variety of tissues, LYVE-1 is still a commonly used marker today to distinguish LECs from other endothelial cells. The goal of this procedure was to isolate LECs from developing mouse embryos with a positive selection approach using dynabeads conjugated to LYVE-1 antibody.

Materials and Reagents

  1. Pipettes tips (Eppendorf, catalog numbers: 022491105 , 022491113 , 022491121 , 022491164 )
  2. Microcentrifuge tube (Fisher Scientific, catalog number: 05-408-130 )
  3. 12-well cell culture plate (Greiner Bio One International, catalog number: 665102 )
  4. 40 μm strainer (Corning, Falcon® , catalog number: 352340 )
  5. Microcentrifuge tube magnet (New England Biolabs, catalog number: S1509S )
  6. Dynabeads: Sheep anti-rat IgG (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11035 )
  7. LYVE-1 antibody, 0.5 mg/ml (R&D Systems, catalog number: AF2125 )
  8. Trizol solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596018 )
  9. RNA Prep Kit (QIAGEN, catalog number: 74106 )
  10. Bovine serum albumin (Rockland Immunochemicals, catalog number: BSA-50 )
  11. PBS w/o Ca2+ and Mg2+ (Merck, Omnipur®, catalog number: 6505-OP )
  12. Collagenase (Sigma-Aldrich, catalog number: 10103578001 )
  13. Dispase (Sigma-Aldrich, catalog number: D4693-1G )
  14. DNase (Thermo Fisher Scientific, AmbionTM, catalog number: AM2224 )
  15. HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14175095 )
  16. Ammonium Chloride (NH4Cl) (Merck, Calbiochem, catalog number: 168320 )
  17. Sodium Bicarbonate (NaHCO3) (Merck, catalog number: SX0320-1 )
  18. Na2EDTA (EDTA disodium) (VWR, catalog number: 97061-022 )
  19. Molecular grade water (Sigma-Aldrich, catalog number: W4502-1L )
  20. PBS/BSA Solution (see Recipes)
  21. Enzyme Solution (see Recipes)
  22. RBC Lysis Buffer Solution (see Recipes)

Equipment

  1. Incubator (Fisher Scientific)
  2. Centrifuge (Eppendorf, model: 5415 R )
  3. NanoDropTM 2000 Spectrophotometers (Thermo Fisher Scientific, model: NanoDropTM 2000 )

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Crosswhite, P. (2018). Isolation of LYVE-1+ Endothelial Cells from Mouse Embryos. Bio-protocol 8(15): e2962. DOI: 10.21769/BioProtoc.2962.

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分类

发育生物学 > 细胞生长和命运决定 > 淋巴管生成

细胞生物学 > 细胞分离和培养 > 细胞分离 > 免疫磁珠

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