EML Erythroid and Neutrophil Differentiation Protocols
骨髓淋巴样红细胞（EML）中的红血球和噬中性细胞分化实验流程
发布: 2014年06月20日第4卷第12期 DOI: 10.21769/BioProtoc.1151 浏览次数: 12698
评审: Lin FangFanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2012
Abstract
Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML cell line is conferred by the presence of a dominant-negative retinoic acid receptor (RAR) originally delivered by retroviral transduction (Tsai et al., 1994), which arrests cells at an early progenitor stage blocked from normal progression into myeloid differentiation. The presence of the RAR trans-gene does not interfere with erythroid differentiation, and it is possible to capture a low percentage of early erythroid, but not myeloid, committed cells in maintenance cultures (Pina et al., 2012; Ye et al., 2005).
Cells can be driven into granulocytic/neutrophil differentiation through the use of high doses of retinoic acid (RA), which overcomes the differentiation block. It should be noted that these pharmacological doses of RA are not compatible with erythroid differentiation, and it is hence not viable to obtain robust erythroid and myeloid differentiation in the same assay. Indeed, colonies scored as mixed-lineage in CFC assays are a mixture of undifferentiated and erythroid cells (Tsai et al., 1994). Nevertheless, robust single-lineage erythroid and neutrophil differentiation can be obtained in liquid culture under defined cytokine conditions, as specified below.
Materials and Reagents
- IMDM (powder or liquid)
- Horse serum (HS) (may require batch testing if low cell viability)
- Foetal bovine serum (FBS)
- L-Glutamine
- Penicillin/Streptomycin (P/S)
- SCF-conditioned medium (SCF-CM) (see Notes)
- Recombinant human erythropoietin (e.g. epoietin alpha, Amgen) (obtained through hospital pharmacy under appropriate local guidelines)
- Recombinant mouse interleukin-3 (IL-3) (e.g. Pepro Tech, catalog number: 213-13 )
- Recombinant mouse granulocyte-monocyte colony-stimulating factor (GM-CSF) (e.g. Pepro Tech, catalog number: 315-03 )
- All-Trans Retinoic Acid (ATRA) (e.g. Sigma-Aldrich, catalog number: 302-79-4 , reconstitute in ethanol)
- Culture supplements and antibiotics (L-Glutamine and Penicillin/Streptomycin)
- Monoclonal anti-mouse antibodies for flow cytometry
- C-kit/CD117 (clone 2B8) (e.g. PE-Cy7, eBioscience, catalog number: 25-1171 ) (suggested use at 1:100 dilution)
- CD34 (clone RAM34) (e.g. Alexa-Fluor 647, eBioscience, catalog number: 51-0341 ) (suggested use at 1:100 dilution)
- Mac-1/CD11b (clone M1/70) (e.g. PE, eBioscience, catalog number: 12-0112 ) (suggested use at 1:100 dilution)
- Gr1/Ly6C (clone RB6-8C5) (e.g. FITC, eBioscience, catalog number: 11-5931 ) (suggested use at 1:100 dilution)
- Sca-1/Ly6A/E (clone D7) (e.g. Pacific blue, BioLegend, catalog number: 122520 ) (suggested use at 1:50 dilution)
Note: Stain on ice, in culture medium, for 20 min; wash with 10-20x volume of medium; pellet cells at 400 x g for 5 min. Re-suspend in 300-500 μl of medium for FACS analysis.
- C-kit/CD117 (clone 2B8) (e.g. PE-Cy7, eBioscience, catalog number: 25-1171 ) (suggested use at 1:100 dilution)
- Trypan blue
Equipment
- T175 tissue culture flasks with filtered cap (for production of conditioned medium)
- T75 or T25 tissue culture flasks with filtered cap, or 6-well plates
Note: For EML cultures, the size of the tissue culture vial is determined by the cell number seeded at the cell densities indicated in the protocol.
- Cell culture incubator
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Pina, C., Fugazza, C. and Enver, T. (2014). EML Erythroid and Neutrophil Differentiation Protocols. Bio-protocol 4(12): e1151. DOI: 10.21769/BioProtoc.1151.
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分类
细胞生物学 > 细胞分离和培养 > 细胞分化
干细胞 > 成体干细胞 > 造血干细胞
干细胞 > 成体干细胞 > 维持和分化
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