| Osteoblast Sorting and Intracellular Staining of CXCL12
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Author:
Date:
2018-05-20
[Abstract] Osteoblasts are bone marrow endosteum-lining niche cells playing important roles in the regulation of hematopoietic stem cells by secreting factors and cell adhesion molecules. Characterization of primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone. Immunophenotyping and flow-based analysis of long bone osteoblasts offer a simplified and rapid approach to characterize osteoblasts. We describe a modified procedure of isolating mouse bone marrow osteoblastic cells based on cell surface immunophenotyping. The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are expressed by osteoblasts and bone marrow stroma cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches ...
[摘要] 成骨细胞是通过分泌因子和细胞粘附分子在调节造血干细胞中发挥重要作用的骨髓内皮细胞生态位细胞。 通过培养胶原酶处理的骨骼的生长已经实现了原代成骨细胞的表征。 长骨成骨细胞的免疫分型和基于流动的分析为表征成骨细胞提供了简化和快速的方法。 我们描述了基于细胞表面免疫分型的分离小鼠骨髓成骨细胞的修改过程。 趋化因子CXCL12（也称为基质衍生因子SDF-1）与其受体CXCR4一起由成骨细胞和骨髓基质细胞表达。 CXCL12-CXCR4轴对于造血干细胞滞留于其生态位（Sugiyama等，2006）和支持白血病启动细胞活性（Pitt等，2015年）。 这里我们描述CXCL12细胞内染色的过程。
【背景】骨髓龛是一种高度组织化的微环境，基质细胞参与与调节HSC静止，分化和动员的造血干细胞（HSC）的直接细胞 - 细胞相互作用（Anthony and Link，2014; Mendelson and Frenette，2014; Morrison和斯卡登，2014年）。 HSC细胞生态位中的多种细胞类型可能以不同的但可能重叠的方式贡献于小生境功能支持。这些细胞包括但不限于成骨细胞，破骨细胞，富含CXCL12的网状（CAR）细胞，Nestin +基质细胞，瘦素受体+（LepR + ...
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| Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells
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Author:
Date:
2018-05-05
[Abstract] Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution ...
[摘要] 骨髓造血干细胞（HSC）需要骨髓微环境来维持和增殖。 骨髓间充质基质细胞（MSC）的培养为体外HSC存活提供适当的环境信号。 在这里，我们提供了描述MSCs培养条件的详细方案，从小鼠骨髓中流式细胞术分离HSCs，并进行称为鹅卵石区域形成细胞（CAFC）分析的MSC和HSC的共培养。 总而言之，CAFC分析可用作高通量体外筛选模型，其中努力了解和开发复杂骨髓疾病的治疗方法。 该方案需要培养MSC，分离LSK细胞（HSC）和执行有限稀释CAFC测定3至4周。
【背景】HSC的增殖，存活和分化潜力非常依赖于其微环境，也被称为小生境。骨髓MSC支持HSC以使其在骨髓龛中保持静止状态。由生态位接收的内在和外在信号有助于将HSC分化为也称为造血的成熟血细胞谱系，而不诱导异常扩增（Yoshihara等人，2007; Spindler等人 ，2014; Hu等人，2016）。鹅卵石区域形成细胞试验（CAFC试验）是长期骨髓HSC和MSC的体外共培养试验。当培养MSC在组织培养皿中完成融合时，将HSC铺在MSC上（de Haan和Ploemacher，2002）。 CAFC测定与骨髓的体内研究相当，并且可用作快速筛选测定以测试HSC的干细胞活性和MSC的支持活性（Ploemacher等人， ...
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| FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles
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Author:
Date:
2015-08-20
[Abstract] Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal ...
[摘要] 荧光活化细胞分选（FACS）是用于从成年小鼠骨骼肌纯化卫星细胞或肌肉干细胞的灵敏和精确的方法（Liu等人，2013; Sacco等人， ，2008; Tierney ，，2014）。 后肢肌肉的机械和酶消化将单核肌细胞释放到悬浮液中。 该方案采用分级分离策略，通过磁性分离和基于FACS的排除，耗尽表达细胞表面标记CD45，CD31，CD11b和Ly-6A/E-Sca1的细胞，并阳性选择表达α7-整联蛋白和CD34的细胞。 这使得研究者能够成功地富集均匀表达配对盒转录因子Pax7并且能够长期自我更新，骨骼肌修复和肌肉干细胞池再增殖的卫星细胞。
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