Name: using an automated cell counter to simplify gene expression studies: sirna knockdown of il-4 dependent gene expression in namalwa cells
Uploaded: 2010-04-14T14:30:00.000+00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells at JoVE.com

1。准备和Namalwa细胞计数<ol><li>从培养瓶中取出的细胞和细胞离心沉淀。删除已知体积的PBS上清和悬浮细胞。在本议定书中所使用的细胞悬浮细胞。如果您使用的是贴壁细胞，你将需要trypsinize之前收集的。 </li><li>要确定活细胞数，染色细胞悬液等分，由0.4％台盼蓝溶液和吸管10μL混合物混合计数幻灯片上1:1，然后插入幻灯片TC10自动细胞计数器（Bio - Rad公司实验室，INC）。 </li><li> TC10细胞计数将自动对焦，以保证最精确的计数，然后使用计数进行。它会自动确定样品中的活细胞和细胞总数的密度。 </li><li>选择查看图像显示viewscreen细胞。然后选择稀释计算器TC10细胞计数，自动计算金额，将需要的细胞混合。 </li><li>本实验使用的是共有3个siRNA的对照siRNA和两个不同的siRNA针对STAT6的基因，加上未转染对照。这就要求在终浓度为5 × 10 6细胞每毫升3.6毫升。 </li><li>输入所需浓度稀释计算器和最终的音量值，并计算出细胞悬液，所需的量的基础上，样品的活细胞计数。另外，手动执行计算。 ，然后转移到一个新的管这些试验的细胞的混合物的要求量。 </li><li>旋转颗粒细胞的细胞混合物，并删除所有的PBS。重悬在3.6毫升基因脉冲电击缓冲液（Bio - Rad公司实验室）。转移到含有适当的siRNA管800μL等份，轻轻混匀。这些细胞是目前电准备。 </li></ol> 2。电穿孔细胞在96孔板<ol><li> 96电板允许复制所有四个一起被电治疗。 </li><li>一个96孔的电板适当的水井，转移到150μL的细胞悬液。 </li><li>插入MXcell电板腔板，并盖上盖子。 </li><li> Electroporate使用的细胞系，并正在使用的缓冲区的优化设置。这些细胞电穿孔使用250伏的仪器设置的指数衰减波，350μF和1000Ω电阻。 </li><li>电完成后，吸管和每口井轻轻地混合，然后将细胞转移到组织培养板中含有预热媒体。 Namalwa细胞是生长在7.5％FCS，1％丙酮酸补充的RPMI（Life Technologies公司）。 </li><li>控制尚未电穿孔的样品取自剩余的细胞悬液，并加入到预热的培养基的培养皿相同的电穿孔细胞。 </li><li>在37℃过夜孵育细胞℃，5％的CO 2。 </li><li> 24小时后，诱导细胞与10 ng / ml的终浓度重组IL - 4（R＆D系统）。控制细胞的第二组只收到其他媒体。所有的细胞都在37 ° C和5％的CO 2孵育一个额外的24小时。 </li></ol> 3。提取和检查的RNA <ol><li>共增长了48小时后，每孔计数细胞，并转移到每口井之一等份为RNA提取的离心管，并冻结了免疫印迹或其他蛋白质分析的第二个等份。 </li><li>样品离心沉淀细胞，然后取出并丢弃上清液从每管。 </li><li>继续与RNA的提取。我们通常使用的奥罗总RNA试剂盒（Bio - Rad公司）根据所包含的协议。 </li><li>当监视的siRNA介导的基因敲除，以验证RNA的质量显得尤为重要，因为降解的RNA会产生误导的结果。 260/280的比例并不表明RNA的完整性，因此重要的是要运行的样品，凝胶或微流体装置，以确认RNA的不退化。 </li><li>为了评估在一个步骤的质量和数量，分析这些样本将被使用的Experion自动电泳系统（BIO - RAD）。 </li><li>首先，热的样品。然后，总理，负载和旋涡的Experion RNA芯片。芯片插入的Experion电泳站，并开始运行。 </li><li>运行完成后，将显示的Experion软件电泳，结果表的形式（即显示RNA浓度，核糖体的比例，和RNA的质量指标数量），和虚拟凝胶结果自动（看起来类似传统的RNA凝胶）。高质量的总RNA样本，通常会有8-10 RQI。 </李&gt; </ol> 4。实时PCR <ol><li> RNA质量核实后，进行cDNA的反应。本实验所用的iScript一步法RT - PCR混合物（BIO - RAD）组合在一个反应​​合成cDNA和实时PCR。 </li><li>设为主结构，包含每个引物RNA模板以外的所有反应组件，并分发到PCR板。 </li><li>一个统一的浓度稀释后的RNA，RNA样品添加适当的水井。每一个反应是成立一式三份。 </li><li>封板，并放入的CFX384实时PCR系统（BIO - RAD）。选择合适的协议，并开始运行。 </li><li>一旦运行完成后，基因表达是通过比较基因表达的基因归参照基因在细胞电穿孔与实验的siRNA细胞电穿孔与对照siRNA正常化的利息确定。 </li></ol> 5。的siRNA敲除分析<ol><li>使用实时定量PCR监测调查中IL - 4 CCL17依赖诱导转录激活因子的作用STAT6的表达CCL17。在这个实验中，被用作参考基因GAPDH的。备用参考基因​​或多个参考基因可以用相同的方式。 </li><li> CFX Manager软件会自动归CCL17表达从同一样品的参考基因表达，并自动比较造成的标准化值，以控制通过简单的选择GAPDH作为“参考”和控制治疗（IL - 4诱导的无控制的siRNA）的治疗CFX Manager软件在实验中设置窗口的“控制”。 </li><li>无IL - 4诱导CCL17的转录低，构水平保持在控制治疗相媲美。 </li><li>诱导的IL - 4和保留正常STAT6通路（即，对照siRNA或未转染对照处理）的细胞表现出8-10倍CCL17表达诱导。 </li><li>与IL - 4，但与STAT6的表达，通过引进任STAT6的siRNA抑制细胞显示，只有约2倍高于非诱导细胞在IL - 4诱导表达的CCL17确认的STAT6作用CCL17表达。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/1904/1904fig1.jpg" alt="图1" /> 图1。 IL - 4信号转导通路的概述 。 的IL - 4及其受体）结合，导致二聚和STAT6的活化。激活STAT6区转移到细胞核，并激活其靶基因，如CC17，转录。 b）在不IL - 4诱导CCL17的转录将保持在低，构水平。 c）在STAT6基因的siRNA介导的沉默，与IL - 4的治疗应该导致很少或根本没有增加CCL17表达。 <img src="/files/ftp_upload/1904/1904fig2.jpg" alt="图2" /> 图2。由IL - 4诱导CCL17测量不同条件下，通过定量实时PCR 。 一）种植无IL - 4的样本低构的表达水平，不论基因的表达。 B）在绿色和蓝色所示，对照样品的反应通常与IL - 4治疗; 8-10倍CCL17诱导。然而，针对STAT6的，在红色和粉色的siRNA转染的细胞，减少了CCL17表达IL - 4，支持这个想法，STAT6起着作用的IL - 4诱导表达的CCL17。

笔者采用Bio - Rad公司实验室产生本文中使用的试剂和仪器。

本文演示了如何electroporate陷入困境的siRNA转染悬浮细胞系，如何遵循这些细胞的基因表达。执行此过程时，重要的是要记得保持尽可能一致的，所有样品的事情。

using an automated cell counter to simplify gene expression studies: sirna knockdown of il-4 dependent gene expression in namalwa cells

使用的siRNA介导的基因敲除继续在基因表达研究中的一个重要工具。 siRNA的研究，不仅要研究单个基因的下调的影响，但也询问信号通路和其他复杂的相互作用网络。这些途径分析需要使用有关的细胞模型和方法，导致少扰动细胞生理学。电穿孔是越来越多地被用来作为一种有效的方式引入难以siRNA和其他核酸，细胞株和原代细胞的转染而不改变正在调查中的信号转导通路。有一个成功的siRNA实验的多个关键步骤，并且有办法在几个实验阶段，同时提高数据的质量，简化工作。为了帮助您与您的siRNA介导的基因敲除项目开始，我们将演示如何执行一个途径的研究，从收集和计数细胞前通过后转实时PCR基因表达分析，以电的完整。下面的研究探讨IL - 4在Burkitt淋巴瘤细胞系（Namalwa）CCL17依赖基因表达的转录激活因子STAT6的作用。本文演示的技术是很有用的一个广泛的siRNA实验的基础上壁和悬浮细胞。我们还将展示如何精简细胞与TC10自动细胞计数计数，如何electroporate MXcell电系统同时使用多个样品，以及如何同时评估RNA的质量和数量的Experion自动电泳系统。

Watch this Scientific Journal Video about Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells at JoVE.com

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

此过程介绍了一个快速简便的工作流程，引入困难的siRNA转染细胞系，并按照实时PCR基因表达。使用自动细胞计数器，多井的电板，全自动电泳站提供快速和可靠的结果，而不需要昂贵的机械手。

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

using-an-automated-cell-counter-to-simplify-gene-expression-studies

Research

JoVE Journal

Biology

15.5K 次观看.  Bio-Rad Laboratories.  中文 

视频: Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

寻找获得性抗靶治疗的驱动途径: 耐药亚克隆生成与基因击倒敏感性恢复

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

科研

癌症研究

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

免疫与感染

Antisense Oligonucleotides as a Tool for Prolonged Knockdown of Nuclear lncRNAs in Human Cell Lines

Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a substantial fraction of lncRNA preferentially accumulates in the nucleus. For analysis of the function of nuclear lncRNAs, it is important to achieve efficient knockdown of these transcripts inside the nucleus. In contrast to the use of RNA interference, a technology that depends on the cytoplasmic silencing machinery, an antisense oligonucleotide (ASO) technology can achieve RNA knockdown by recruiting RNase H to the RNA-DNA duplexes for nuclear RNA cleavage. Unlike the use of CRISPR-Cas tools for genome engineering, where possible alterations in the chromatin state can occur, ASOs allow the efficient knockdown of nuclear transcripts without modifying the genome. Nevertheless, one of the major obstacles to ASO-mediated knockdown is its transitory effect. For the study of long-lasting effects of lncRNA silencing, maintaining efficient knockdown for a long time is needed. In this study, a protocol was developed to achieve a knockdown effect for over 21 days. The purpose was to evaluate the cis-regulatory effects of lncRNA knockdown on the adjacent coding gene RFC4, which is related to chromosomal instability, a condition that is observed only through time and cell aging. Two different human cell lines were used: PrEC, normal primary prostate epithelial cells, and HCT116, an epithelial cell line isolated from colorectal carcinoma, achieving successful knockdown in the assayed cell lines.

To analyze the function of lncRNAs in time-dependent processes such as chromosomal instability, a prolonged knockdown effect must be achieved. To that purpose, presented here is a protocol that uses modified antisense oligonucleotides to achieve effective knockdown in cell lines for 21 days.

Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a ...

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

使用微流体装置生产siRNA负载的脂质纳米颗粒

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

生物化学

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

生物学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...