| Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model
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Author:
Date:
2021-03-20
[Abstract] The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development. These qualities become important for in vivo cell specific depletions so that the elimination of the targeted cells can be tracked and validated in real time through microscopy. Multiple methods to deplete macrophages in zebrafish are available, including genetic (such as an irf8 knockout), chemogenetic (such as the nitroreductase/metronidazole system), and toxin-based depletions (such as using clodronate liposomes). The use of clodronate-containing ...
[摘要] [摘要]为了进行能力在体内巨噬细胞比耗尽仍然是有效的手段，在广泛的生理环境的巨噬细胞的揭开功能。与鼠模型相比，斑马鱼具有良好的成像能力，因为它们的光学透明性从单细胞阶段到整个幼体发育期开始。这些素质成为重要的体内细胞特异的枯竭，使靶细胞的消除可以跟踪并通过显微镜进行实时验证。多种方法以耗尽巨噬细胞在斑马鱼中是可用的，包括遗传（如IRF8敲除），chemogenetic（如在 硝基还原酶/甲硝唑系统）和基于毒素的消耗（例如使用氯膦酸盐脂质体）。在吞噬脂质体后，使用含氯膦酸盐的脂质体诱导巨噬细胞凋亡可有效地消耗巨噬细胞并测试其吞噬能力。在这里，我们描述了通过静脉注射补充有荧光右旋糖酐共轭物的脂质体氯膦酸盐对斑马鱼幼虫体内巨噬细胞进行系统耗竭的详细方案。与荧光右旋糖酐共注射可以实时跟踪巨噬细胞耗竭情况，从验证静脉内注射成功到摄取巨噬细胞分子及其最终死亡开始。为了验证高度的巨噬细胞耗竭，可以在幼虫早期进行氯膦酸盐注射时通过快速中性红色活体染料染色来确定脑巨噬细胞（小胶质细胞）的清除水平。
图形概要：
幼虫斑马鱼脂质体氯膦酸盐体内巨噬细胞特异性清除的实验工作流程
[背景]巨噬细胞是先天免疫系统的关键组成部分，并响应于感染，无菌性炎症，与环境变化中发挥重要作用。使巨噬细胞功能与不同生理环境中相互作用细胞类型的复杂混合物脱钩的最有效方法之一是能够特异性消除巨噬细胞并分析表型结果。小鼠中的此类耗竭实验为巨噬细胞的作用提供了很多见识（Hua等人，2018; ...
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| Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs
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Author:
Date:
2017-06-05
[Abstract] Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup ...
[摘要] 与qPCR（qChIP）或高通量测序（ChIP-Seq）相结合的染色质免疫沉淀已被广泛用于识别转录因子的DNA结合位点或基因组中各种组蛋白标记的定位。 ChIP实验通常包括7个步骤：收集生物样品（A），交联蛋白质到DNA（B），染色质分离和通过超声处理分离（C），超声处理测试（D），用针对蛋白质的抗体进行免疫沉淀感兴趣的组蛋白标记（E），DNA回收（E），通过PCR或测序鉴定因子相关DNA序列（F）。这里描述的协议可以容易地用于ChIP-seq和ChIP-qPCR实验。描述在完整的果蝇组织中优化分析的实验设置条件的整个过程可以在四天内完成。
背景 尽管永生化的培养细胞广泛用于研究各种细胞类型的染色质景观，但是在生理条件下在体内探测相互作用的有价值的方法对于进行转录的时间或空间比较分析是必要的因子和组蛋白修饰图在不同阶段的果蝇发展或不同组织之间。在这里，我们提供了一个详细的ChIP协议，已被优化，以便在整个果蝇胚胎和幼虫成像光盘上工作，突出关键的实验参数。
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| Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
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Author:
Date:
2016-07-20
[Abstract] Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the ...
[摘要] 无机多磷酸盐（polyP）是存在于原核和真核生物中的线性聚合物，由通过磷酸酐键连接的三至数百个正磷酸盐残基制成。 这种分子的生物学作用超越了作为P 1储存或能量源以代替ATP。 例如，在酵母中，polyP水平与应激适应相关，并且该分子已经显示为蛋白质多磷酸化的底物。 在这里，我们描述了从酵母酿酒酵母中纯化polyP的两种不同方法和随后的通过分光光度法测量在酶水解纯化的polyP时产生的Pi来定量polyP水平的方案。 必须注意的是，所使用的纯化方案极大地影响所获得的polyP值。
图1. polyP的酶水解
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