Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine
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Author:
Date:
2016-12-20
[Abstract] RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila intestinal cells (Chen et al., 2016), in which RNA amplification is not required.
[摘要] RNA测序(RNA-seq)已经成为描述基因表达的流行方法。 在许多应用中,一个共同目的是在不同的生物学或病理学条件下鉴定差异表达的基因和途径。 该方案提供了〜250,000个分选的果蝇肠细胞的RNA-seq分析的详细程序(Chen等,2016),其中不需要RNA扩增。 【背景】RNA-seq的转录组分析已经成为鉴定不同生物或病理条件下差异表达基因和途径的常用方法。 对于产生低mRNA水平的样品,通常在深度测序之前进行RNA或cDNA扩增(Dutta等,2015)。 然而,这个程序可能会潜在地省略以低丰度表达的重要候选人。 在这里,我们提供了不需要RNA扩增的分选的果蝇肠细胞的RNA-seq分析的详细程序。
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Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation
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Author:
Date:
2016-11-05
[Abstract] The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation.
The protocol can be divided into four main steps: preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement. This assay is limited to manual or semiautomatic screening, and can be used in an efficient ...
[摘要] Limonium 已知有性和无融合生殖(通过种子的无性繁殖)繁殖模式。在这里,我们提出解剖协议为胚珠的发芽。使用微分干涉对比(DIC)显微镜。该协议允许更好地处理胚珠,并提供优于早期技术,特别是在较大胚珠的某些优势。这种方法还能够观察到完整胚珠中的减数分裂和胚囊发育,以及容易检测到区别性和无序性发育的事件。 [背景] 发生在胚珠发育期间,有必要细胞学检查胚珠。这项研究可以涉及显微镜观察石蜡或树脂包埋,切片材料或清除的器官。在D'Amato(1940; 1949)的先驱作品中公开了对性和无融合生殖物种中胚珠和胚囊发育的第一次细胞学研究。在这些作品中,花使用Karpechenko的方法固定,包埋在石蜡中,切片并用Heidenhain的铁苏木精染色,其染色质和染色体在细胞核中染色。使用这些方法的花芽切片可导致由于单个细胞的部分破坏结构完整性而具有差质量的制备物。更容易的选择是清除福尔马林:乙酸:乙醇固定的器官并用纯的Mayer's hemalum染色(Wallis,1957; Stelly等人,1984)。这种技术需要少得多的时间和劳动,特别是对于通常在卵巢中形成小胚珠的物种,这是 Limonium ...
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ELISA Measurement of Mouse IL-2
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Author:
Date:
2012-01-05
[Abstract] Interleukin-2 (IL-2) is a cytokine secreted by T cells that is essential for immune system activation. This protocol is routinely used for quantification of IL-2 concentration in the supernant of cultured lymphocytes under various stimulations and co-culturing conditions. Upon slightly modification and optimization, this protocol can also be adapted to quantitatively measure other secreted proteins and bio-molecules.
[摘要] 白细胞介素-2(IL-2)是由免疫系统激活所必需的T细胞分泌的细胞因子。 该方案常规用于在各种刺激和共培养条件下定量培养的淋巴细胞的上清液中的IL-2浓度。 在稍微修改和优化后,该方案也可以适用于定量测量其他分泌的蛋白质和生物分子
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