Limiting Dilution Assays to Determine Frequencies of Lymphohematopoietic Progenitors
有限稀释法测定淋巴造血祖细胞的比例
发布: 2014年02月20日第4卷第4期 DOI: 10.21769/BioProtoc.1045 浏览次数: 9840
评审: Omar AkilAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2013
Abstract
This protocol is useful to determine the frequencies of lymphohematopoietic progenitors in tested samples. To effectively support the growth and differentiation of primitive lymphohematopoietic progenitors, complex signals from stromal cells are important. Several stromal cell lines are known to support both lymphoid and myeloid cells simultaneously in mouse. In this protocol, we introduce two stromal co-culture systems for murine lymphohematopoietic progenitors and their application for limiting dilution assays.
Materials and Reagents
- Stromal cells (MS5 cells or OP9 cells)
- Growth factors
a. rm SCF (10 ng/ml) (e.g. R&D systems, catalog number: 455-MC-010 )
b. rm Flt3-ligand (20 ng/ml) (e.g. R&D systems, catalog number: 427-FL-025 )
c. rm IL-7 (1 ng/ml) (e.g. R&D systems, catalog number: 407-ML-005 )
- Antibodies
a. Anti-mouse Mac1 (BD Biosciences, catalog number: 557396 )
b. Gr1 (BD Biosciences, catalog number: 553128 )
c. CD19 (BD Biosciences, catalog number: 557399 )
d. CD45R/B200 (BD Biosciences, catalog number: 553092 )
e. CD45 (BD Biosciences, catalog number: 550994 )
- Trypsin/EDTA solution (Nacalai Tesque, catalog number: 35554-64 )
- EDTA solution (Nacalai Tesque, catalog number: 14367-74 )
- Alpha Modification of Eagle’s Medium (Corning, cellgro®, catalog number: 50-012 )
- Minimum Essential Medium Alpha Medium (Gibco®, catalog number: 41061-029 )
- FCS (MP Biomedicals, catalog number: 2916754 )
- L-glutamine (Corning, cellgro®, catalog number: 61-030 )
- Penicillin-Streptomycin (Nacalai Tesque, catalog number: 26253-84 )
- MS-5 growth media (see Recipes)
- OP-9 growth media (see Recipes)
Equipment
- Cell sorting machines (BD Biosciences, FACSAriaTM) with the automated cell deposition system
- 96-well flat bottom plates
- 10 cm plastic dishes
- CO2 incubators
- Flow cytometry system
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yokota, T. and Satoh, Y. (2014). Limiting Dilution Assays to Determine Frequencies of Lymphohematopoietic Progenitors. Bio-protocol 4(4): e1045. DOI: 10.21769/BioProtoc.1045.
Download Citation in RIS Format
分类
干细胞 > 成体干细胞 > 基质细胞
免疫学 > 免疫细胞分离 > 淋巴细胞
免疫学 > 免疫细胞分离 > 维持和分化
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