| 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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| Methods for Detecting Microbial Methane Production and Consumption by Gas Chromatography
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Author:
Date:
2016-04-05
[Abstract] Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane ...
[摘要] 甲烷是能量密集的燃料，但是也是比CO 2更加不利于环境25倍的温室气体。 甲烷可以通过蛇形化，化学方式通过Sabatier或Fisher-Tropsh化学，或通过微生物生物地生产（Berndt等人，1996; Horita和Berndt，1999; Dry，2002; Wolfe， Thauer，1998; Metcalf>等人，2002）。 甲烷生物是通过产生甲烷气体作为代谢副产物而生长的厌氧古生菌（Wolfe，1982; Thauer，1998）。 我们的实验室已经开发和优化了三种不同的气相色谱仪利用测定法来表征甲烷菌代谢（Catlett等人，2015）。 在这里我们描述的终点和动力学测定可用于测量甲烷产生甲烷生物或甲烷营养微生物的甲烷消耗。 该方案可用于测量甲烷产生或通过微生物纯培养物或通过富集培养物消耗。
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| Isolation and Purification of Murine Microglial Cells for Flow Cytometry
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Author:
Date:
2016-01-05
[Abstract] The detailed protocol is used to isolate different cell types from murine brain as glial cells, including microglia, using an enzymatic digestion that minimizes cellular mortality. A Percoll gradient (30% to 80%) separation allows a maximal recovery of isolated murine microglial cells prior to flow cytometry analysis.
[摘要] 详细的协议是用于分离不同的细胞类型从鼠脑作为神经胶质细胞，包括小胶质细胞，使用最小化细胞死亡率的酶消化。 Percoll梯度（30％至80％）分离允许在流式细胞术分析之前分离的小鼠胶质细胞的最大恢复。
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