Competitive Bone-marrow Transplantations
竞争性骨髓移植
发布: 2014年06月05日第4卷第11期 DOI: 10.21769/BioProtoc.1145 浏览次数: 25642
评审: Lin FangFanglian HeAnonymous reviewer(s)
PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
May 2012
Abstract
Competitive bone marrow transplantation assay measures multi-lineage reconstitution of hematopoiesis in irradiated transplant recipient mice. Thus this assay is routinely used to determine haematopoietic stem and progenitor cells (HSPCs) functionality in vivo. The principle of the method is to transplant bone marrow donor cells (derived from transgenic mice of choice) on C57BL6 background together with normal competitor bone marrow. In order to distinguish donor from competitor cells upon transplantation, usually competitor mice are congenic and carry the differential B cell antigen originally designated Ly5.1 and CD45.1.
A typical competitive bone marrow transplantation experiment will contain two transplantation groups, donor (transgenic mice of choice and their controls) are transplanted in competition with normal competitors and engraftment efficiency is evaluated in both blood and bone marrow.
Hematopoesis (造血)
HSCs (造血干细胞)
Competitive (竞争的)
Stem cell engrafment (干细胞植活)
Materials and Reagents
- C57BL6 (CD45.2) mice (bred in our laboratory)
- C57BL6 (CD45.1) mice (bred in our laboratory)
- Ciprofloxacin 200 (200 mg/100 ml Bayer)
- Phosphate Buffered Saline (PBS) (without Ca2+ and Mg2+) (Sigma-Aldrich, catalog number: D8537 )
- Turk’s solution (Merck KGaA, catalog number: 1.09277.0100 )
- Heparin sodium salt (Sigma-Aldrich, catalog number: H3393-100KU )
- Ficoll-Paque plus (GE Healthcare, catalog number: 17-1440-03 )
- FITC anti-mouse CD45.1 (cloneA20) (Biolegend, catalog number: 110705 )
- PE anti-mouse CD115 (clone AFS98) (Biolegend, catalog number: 135506 )
- APC anti-mouse B220 (clone RA3-6B) (eBioscience, catalog number: 17-0552-81 )
- FITC anti-mouse Gr-1 (clone RB6-8C5) (Biolegend, catalog number: 108405 )
- APC anti-mouse Ter119 (clone TER-119) (Biolegend, catalog number: 116211 )
- PE anti-mouse CD3 (clone 145- 2C11) (Biolegend, catalog number: 100307 )
- APC anti-mouse NK1.1 (clone PK136) (Biolegend, catalog number: 108709 )
- Streptavidin PerCp (Biolegend, catalog number: 405213 )
- Sodium azide
- FACS buffer (see Recipes)
- RBC lysis buffer (see Recipes)
- Heparin solution (see Recipes)
Equipment
- 3 ml syringe (BD Biosciences, catalog number: 305270 )
- Forceps and sharp scissors
- Needles (21 G x 1.5-Nr.2 0.8 mm x 40 mm) (BD Biosciences, catalog number: 304432 )
- 0.5 ml syringes (BD, catalog number: 320933 )
- Mouse restrainer
- Red light lamp
- Tissue culture (TC) dish (6 well plate) (Corning, catalog number: 3516 )
- 15 ml conical tubes (BD Biosciences, Falcon®, catalog number: 352196 )
- 70 μM nylon strainers (BD Biosciences, Falcon®, catalog number: 352350 )
- Inverted microscope
- Ice bucket
- Hemacytometer (Sigma-Aldrich, catalog number: Z359629-1EA )
- Centrifuge
- Scalpel knife
- Eppendorf tubes (Axygen®, catalog number: MCT-175-C ).
- FACS micro-tubes (USA Scientific, catalog number: 1412-1000 )
- FACS tubes (BD, catalog number: 352054 )
- Mice Irradiation machine (Gamma cell 40 Exactor) (InsideView)
- BD LSR II flow cytometer
Software
- FACSDiva software
- FlowJo software
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Maryanovich, M. and Gross, A. (2014). Competitive Bone-marrow Transplantations. Bio-protocol 4(11): e1145. DOI: 10.21769/BioProtoc.1145.
Download Citation in RIS Format
分类
干细胞 > 成体干细胞 > 造血干细胞
免疫学 > 动物模型 > 小鼠
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
