| Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism
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Author:
Date:
2018-09-05
[Abstract] Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery.
The ...
[摘要] 使用气相色谱质谱（GC-MS）分析柠檬酸循环（CAC）和相关中间体（如谷氨酸，谷氨酰胺，GABA和天冬氨酸）是一种分析方法，用于识别明显相关和不相关的能量途径之间的意外相关性代谢。通过使用已知量的样品添加的参考标准，中间体可以表示为它们的绝对浓度或相对比例。 GC-MS还可以区分重标记分子（ 2 H-或 13 C-标记的）和天然存在的最丰富的分子。使用示踪剂的应用还可以评估在各种生理和病理条件下以及用于途径发现的特定代谢池的周转。
以下方案是一种相对简单的方法，不仅对小浓度的代谢中间体敏感，而且还可以 in vivo 或 in vitro 用于确定各种新陈代谢的完整性途径，如特定代谢物池内的通量变化。我们使用该协议来确定磷酸烯醇丙酮酸羧激酶1 （ Pck1 ）基因在小鼠巨噬细胞中的作用，以确定 13 C将葡萄糖标记为特定的CAC代谢物库
【背景】随着细胞和小鼠中基因表达改变的发展，需要了解这些缺失或过表达的基因如何影响代谢途径的调节。在该方案中，我们使用稳定同位素来确定进入CAC的葡萄糖通量如何改变葡萄糖对柠檬酸盐，琥珀酸盐和苹果酸盐的贡献。使用稳定同位素和目标分析代谢只是在细胞培养中使用稳定同位素的一个好处。
本方案中描述的用于细胞内代谢物功能定量的方法是通过在U- 13 ...
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| Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina
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Author:
Date:
2017-02-05
[Abstract] All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During ...
[摘要] 构成成熟视网膜的所有七种视网膜细胞类型都是由普通的多能视网膜祖细胞池（RPC）产生的（Wallace，2011）。已经产生足够数量的特定细胞类型的RPC知道的一种方式是通过负反馈信号，其由分化细胞发射并且必须达到阈值水平以阻止该细胞类型的额外分化。评估负反馈信号是否由分化细胞发出的关键测定是异源沉淀测定，其中早期RPC被解离并用BrdU标记，然后与20倍过量的解离的分化细胞混合。然后将组合的细胞再次聚集并在细胞膜上培养7-10天。在这段时间内，RPC将会分化，BrdU + RPC的命运可以使用细胞类型特异性标记进行评估。开发这种沉淀测定的研究人员最初表明，当两种细胞类型混合在一起时，新生儿RPC与胚胎RPC相比，在加速进度条件下产生杆（Watanabe和Raff，1990; Watanabe等，1997）。我们已经使用这种测定来证明我们发现作为视网膜神经节细胞（RGC）分化的负调节物的声刺猬（Shh）促进RPC增殖（Jensen和Wallace，1997; Ringuette等，2014）。最近我们修改了异质性沉淀测定法，以评估视网膜无长突细胞的反馈信号的作用，将转化生长因子β2（Tgfβ2）鉴定为负反馈信号，并将Pten作为Tgfβ2应答的调节剂（Ma et al。，2007 ; ...
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