Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood
从人胎肝和脐带血中分离CD34+细胞
发布: 2013年12月05日第3卷第23期 DOI: 10.21769/BioProtoc.991 浏览次数: 14693
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2013
Abstract
CD34 is a glycosylated cell surface protein and represents a well-known marker for primitive progenitor cells in various organs, especially cord blood, bone marrow and fetal liver. CD34+ progenitor cells are suitable for a series of studies, e.g. cell differentiation, transplantation as well as construction of humanized mouse models. Here, we describe a method to isolate CD34+ cells from the human cord blood and fetal liver.
Cord Blood (脐带血)
Fetal liver (胎肝)
Magnetic purification (磁净化)
Hepatic progenitor cells (肝前体细胞)
Materials and Reagents
- Collagenase, Type IV (Life Technologies, catalog number: 17104019 )
- Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
- ACK Lysing Buffer (Life Technologies, catalog number: A1049201 )
- RoboSepTM Buffer (STEMCELL Technologies, catalog number: 20104 )
- Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
- EasySepTM Human Cord Blood CD34 Positive Selection Kit (STEMCELL Technologies, catalog number: 18096 )
- StemSpanTM SFEM (STEMCELL Technologies, catalog number: 0 9650 )
- DMSO Hybri-Max Sterilefilt (Sigma-Aldrich, catalog number: D2650 )
- Hyclone Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 30070.03 )
- Liquid nitrogen
- 1 mg/ml Collagenase IV (see Recipes)
- Freezing medium (see Recipes)
- PBS/EDTA (2 mM) (see Recipes)
Equipment
- Syringe Filter (0.20 μm Blue Rim) (Minisart®, catalog number: 16534-K )
- Centrifuge Tube (50 ml Blue Cap) (BD Biosciences, Falcon®, catalog number: 35 2070 )
- T75 cell culture flask
- Petri dish (90 x 15 mm) (Thermo Fisher Scientific, catalog number: BSN 101VR20 )
- Cell scrapers (L29 cm Blade 2 cm) (SPL Life Sciences Co., catalog number: 90030 )
- Shaker Incubator (New Brunswick Scientific, model: Innova 4000 )
- Sterile wire mesh (100 μm) (cut larger than the size of a 90 x 15 mm Petri dish)
- Syringe (10 cc Luer Lok) (BD, catalog number: DS-BD-15026C )
- Tabletop Centrifuge Legend RT (Sorvall)
- FalconTM Polystyrene Round-Bottom Tubes (14 ml) (BD, catalog number: 352057 )
- Big EasySep® Magnet (STEMCELL Technologies, catalog number: 18001 )
- Hematocytometer (TOMY DIGITAL BIOLOGY, catalog number: DHC-N01 )
- Cryovial
- Cryo tube (free standing 2 ml) (Corning Incorporated, catalog number: 430488 )
- Mr. Frosty Freezing Container (Cyro 1DEGC) (Thermo Fisher Scientific, catalog number: PLW- FS-00033 )
- Leucosep tube
- 37 °C incubator
- Centrifuge
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Chen, Q. and Chen, J. (2013). Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood. Bio-protocol 3(23): e991. DOI: 10.21769/BioProtoc.991.
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分类
细胞生物学 > 细胞分离和培养 > 细胞分离
干细胞 > 成体干细胞 > 造血干细胞
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