作者承认下洛佩兹和 Miren Telletxea 为视频录制和编辑提供的帮助。ACR 由 Ikerbasque 奖学金和协会 de Celiacos 马德里 (ACM) 的研究项目资助。JRB 由 ISCIII-PI16/00258 项目资助, 并由欧洲联盟 ERDF/英法基金会共同出资, "成为欧洲的一种方式"。是由巴斯克卫生部的一个研究项目赠款2015111068资助的。IRG 和 AJM 分别得到 UPV/EHU 和巴斯克教育部 predoctoral 奖学金资助。

这项研究获得了该大学医院伦理委员会的批准, 并在获得所有受试者或其父母的知情同意后进行分析。
1. 活检收集
注: 患者远端十二指肠活检标本可在常规诊断内窥镜下进行, 检查范围为2至10毫克。活检组织是由不同细胞类型组成的复杂组织, 包括上皮、免疫和内皮细胞。临床肠胃根据既定的临床指南执行内窥镜。
<ol><li>在内窥镜下, 将活检放在无菌滤纸上。</li>
	<li>使用镊子转移活检1.5 毫升管, 浸泡在 PBS, 并将其放入 cryotube。</li>
	<li>把管子放在冰上直到他们到达实验室。<ol>
<li>立即开始处理新鲜的活检。</li>
	</ol>
</li>
	<li>对于冰冻活检, 闪光冷冻的活检包含管和贮存在液氮, 直到使用。 注意: 在添加了冷补充的缓冲1之前, 保持样品冻结是很重要的。</li>
</ol>2. 缓冲准备
注意: 开始之前, 请按如下所示补充缓冲区。
<ol><li>将1毫米的 (最终浓度) 和蛋白酶和磷酸酶抑制剂鸡尾酒 (1x 最终浓度) 添加到低渗缓冲1和高渗缓冲 2 (表 1)。<ol>
<li>准备1.5 毫升的缓冲区1和150µL 的缓冲区2每个样本。德勤将防止样品的氧化。</li>
	</ol>
</li>
	<li>通过加入1% 洗涤剂 (壬 phenoxypolyethoxylethanol) 来制备核洗涤缓冲器, 最后浓度为0.1% 到已经补充的缓冲1。每个样品准备1毫升的原子核洗涤缓冲器。</li>
</ol>3. 同质活检
<ol><li>
新鲜活检
	<ol>
<li>把一次性杵放在电动搅拌机里。确保杵达到管的底部。</li>
		<li>从 cryotube 中取出活检, 把它放在一个1.5 毫升的试管里, 用吸管尖。</li>
		<li>添加200µL 的补充缓冲1到1.5 毫升管。</li>
		<li>融汇活检与杵, 直到组织完全中断, 并保持在冰上。</li>
		<li>一旦所有的活检都是均匀的进行步骤4。 注意: 一次性杵必须在活检之间改变。</li>
	</ol>
</li>
	<li>
冰冻活检 注意: 小心处理液氮;与皮肤或眼睛接触液氮可能导致严重的冰冻损伤。使用液氮时, 要始终保护双手和眼睛。<ol>
<li>在盒子里取一些液氮储存从氮气罐中回收的 cryotubes。</li>
		<li>在氮气罐中找到活检, 并将 cryotube 放在盒子里, 用液氮。对所有活检重复此步骤。</li>
		<li>将活检从 cryotube 转移到1.5 毫升管。 注: 活检将附在冷冻管上。用一根不育的吸管来推活检, 这样它就会从管子里分离出来。</li>
		<li>添加200µL 的补充缓冲1。</li>
		<li>融汇组织使用杵和砂浆, 直到组织完全中断, 保持温度在4摄氏度整个过程。 注意: 在添加缓冲区之前不要让活检解冻。</li>
	</ol>
</li>
</ol>4. 细胞质分离
<ol><li>在冰上孵化10分钟的匀质活检。</li>
	<li>添加10µL 1% 洗涤剂 (壬 phenoxypolyethoxylethanol) 到每个样品到最后浓度为0.05%。 注意: 由于温和的洗涤剂会扰乱细胞膜, 细胞会膨胀并通过渗透裂解而爆裂。</li>
	<li>在冰上孵化5分钟。</li>
	<li>漩涡简要地和离心机在 400 x g 在4˚C 为2分钟。</li>
	<li>涡流后, 取出上清, 并将其转移到新的清洁1.5 毫升管;这将是细胞质分数。不要丢弃小球。</li>
</ol>5. 核隔离
<ol><li>并用重悬用200µL 核洗涤缓冲器从上一步对颗粒进行了处理。</li>
	<li>离心管为2分钟在 400 x g 和4°c。</li>
	<li>放弃上清, 再重复洗涤程序两次。 注: 洗涤将去除核分数中的细胞质污染。</li>
	<li>第三次清洗后, 并用重悬在100µL 2 的缓冲颗粒。</li>
	<li>用力摇动样品4˚C 30 分钟. 或者, 每5分钟在冰和旋涡上孵化样品。</li>
	<li>震动后, 在最高速度 (> 1.2万 x g) 和4摄氏度离心样品10分钟。</li>
	<li>涡流后, 将上清液转移到新的清洁1.5。毫升管。这将是核分数。</li>
</ol>6. 蛋白质量化
注: 用二辛可宁酸 (蛋白质检测试剂盒) 量化蛋白质。蛋白质在分数中的浓度将介于0.5 和5µg/µL 之间, 在核分数中较低 (见表 2为由2毫克活检结果产生的蛋白质浓度)。
<ol><li>用以下牛血清白蛋白 (BSA) 量 (µg)12: 0-0. 5-1-2-3-4-5-6, 制备标准曲线。</li>
	<li>准备所需的试剂, 以使用100µL 的最终可达性的组合, 每个样本。</li>
	<li>吸管2µL 的每一个蛋白质分数在一个96井板, 添加的综合体的组合, 并按照制造商的说明, 为阅读。</li>
</ol>

<ol>
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R., Ronald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in disease investigating the gastrointestinal tract. , (1998).</li><li>Alex, P., Gucek, M., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+proteomics+in+the+study+of+inflammatory+bowel+diseases:+Current+status+and+future+directions+with+available+technologies.">Applications of proteomics in the study of inflammatory bowel diseases: Current status and future directions with available technologies.</a> Inflammatory bowel diseases. 15 (4), 616-629 (2009).</li><li>Ericsson, C., Franz&#233;n, B., Nist&#233;r, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frozen+tissue+biobanks.+Tissue+handling,+cryopreservation,+extraction,+and+use+for+proteomic+analysis.">Frozen tissue biobanks. 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作者没有什么可透露的。

这里描述的协议用于肠道活检的核和细胞质分馏。纯化的蛋白质不变性, 不仅可以用于西方印迹分析如图 2和3 , 但也在化验要求本机折叠的蛋白质, 如免疫沉淀, 电泳移动转移检测 (EMSA) 或本机聚丙烯酰胺凝胶电泳 (页)。
所述方法依赖于组织的机械均匀化和渗透裂解, 在不影响核膜的情况下爆裂细胞膜。其结果是将用于下游分析的细胞质和核?...

蛋白质参与细胞内几乎所有的生物功能, 其结构、数量或位置的任何变化都可能导致致病性的情况。组织样品分馏法是降低疾病相关蛋白质分析复杂性的有效方法。一些研究使用蛋白质定位信息或丰富一个特定细胞室的蛋白质, 因此, 一个完整的蛋白质可再生分馏的协议是有用的回答某些生物学问题。确定蛋白质的亚细胞定位和监测其在基础和疾病条件下的区划再分配或相互作用, 将有助于确定与疾病相关的功能差异1,2。因此, 该方法的目的是重现性分级小肠组织活检, 无论是新鲜和冷冻, 进入细胞质和核亚细胞室。
肠道活检标本通常获得在内窥镜程序3 (图 1), 可以有效地用于蛋白质定量或免疫沉淀研究。由于肠道上皮组织样本将含有可能直接参与疾病发病机制4的蛋白质, 肠道活检标本是成功鉴别肠道疾病特异性的一个非常有价值的来源。蛋白质。储存冷冻, 病人组织样本连同临床信息是蛋白质分析的有用资源, 一个简单和可重复的样品准备是提供细胞区划信息的关键问题, 使用限制数量的冷冻组织5。
有几个商业可用的工具包分离的细胞分数, 但这些是更昂贵的, 一般比这里提出的协议花费更多的时间。基于多级梯度离心和连续梯度的协议以前也被用于不同组织6、7中各种细胞间隔的分馏。但是, 保持连续渐变的一致性往往是一项艰巨的任务。先前已经描述了基于连续裂解膜的类似协议, 但通常需要两个以上的缓冲器, 而手的时间更长8 。
当分馏组织样品时, 请记住, 组织样本呈现细胞细胞和细胞基质相互作用, 它们既不存在于培养细胞中, 又在蛋白质提取过程中很重要。在不影响蛋白质质量的情况下, 细胞与胞外基质的适当分解将是肠道组织蛋白分离9的关键因素。在这个协议中, 细胞细胞和细胞矩阵的接触首先被打破, 释放细胞并允许缓冲区到达各个细胞。为了避免在分馏之前进行蛋白质间的混合, 必须在不改变细胞或核膜完整性的情况下进行接触击穿。
由于肠道黏膜10中各种蛋白酶的丰富, 在提取过程中控制蛋白质降解具有重要意义。为了尽量减少潜在的蛋白质降解, 必须将多种蛋白酶抑制剂包括在蛋白质提取缓冲器中。此外, 如果提取物将用于功能化验, 这是必要的, 以避免蛋白质变性或蛋白水解, 因为这将导致蛋白质活性的损失11。为此, 该协议的执行在4摄氏度, 新鲜的 phenylmethylsulfonyl 氟化物 (PMSF) 被添加到缓冲区在最后浓度为0.1 毫米之前, 使用进一步抑制蛋白质水解。
细胞分馏的第一步是组织破坏和细胞裂解。如前所述, 其目的是将细胞分解, 并以最小的损伤打开。肠道组织样品必须均匀化, 细胞裂解以达到最大的细胞膜断裂。在本协议中, 我们使用电动杵式搅拌机打破了在几秒钟内均匀化的肠道组织通过高速涡流行动。均匀化后, 组织细胞在低渗缓冲中孵化, 它会爆裂细胞膜, 但会使细胞核保持完好, 其次是添加非变性洗涤剂 (壬 phenoxypolyethoxylethanol) 和短涡来分离细胞核。从细胞质分数。胞浆切除后, 细胞细胞核膜爆裂成高渗缓冲液, 晃动。
本文提出的方法是在内镜手术中获得的新鲜和冰冻小肠活检的亚细胞分馏的适当方法, 在2至10毫克的重量范围内。该协议简单、重现性好, 可在一小时内使用基本的实验室设备和试剂进行。

<table><tbody><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034-1kg</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333-1kg</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E9884-100G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>5588886-1kg</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>10197777001</td><td></td></tr><tr><td>PMSF</td><td>Thermo Scientific</td><td>36978</td><td></td></tr><tr><td>Proteinase and phosphatase inhibitors</td><td>Thermo Scientific</td><td>A32959</td><td></td></tr><tr><td>NP-40&amp;nbsp;</td><td>Sigma Aldrich</td><td>CA-630</td><td>Detergent</td></tr><tr><td>BCA assay</td><td>Thermo Scientific</td><td>23227</td><td>Protein quantification kit</td></tr><tr><td>Disposable plastic pestles</td><td>Sigma Aldrich</td><td>Z359947-100EA</td><td></td></tr><tr><td>Dounce homogeneizer</td><td>VWR</td><td>431-0100</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415-R</td><td></td></tr><tr><td>Shacker</td><td>IKA</td><td>MS3 basic</td><td></td></tr></tbody></table>

subcellular fractionation from fresh and frozen gastrointestinal specimens

本议定书的目的是分级人肠道组织获得的内窥镜进入核和细胞质室, 以定位分析特定的蛋白质或蛋白质复合体在不同的组织状态 (即,健康与疾病)。该方法对新鲜和冷冻小肠组织标本的分馏有重要意义;它是容易接近的所有实验室, 而不是费时。

图 1显示了一个具有完整绒毛和隐窝结构的小肠活检切片的苏木精的伊红染色。
使用本议定书的核和细胞质分馏的代表性结果见图 2和3。在图 2所示的实验中, 新的 (Fh) 和冰冻的 (组织) 活检是在这里提出的协议同时进行分割的, 并使用每个样本的20微克进行?...

Watch this Scientific Journal Video about Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens at JoVE.com

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

在这里, 我们提出了一个协议, 以执行一个简单的细胞分离的胞质和核蛋白在人类新鲜和冰冻肠道活检。

新鲜冷冻胃肠标本的亚细胞分离

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

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An Overview of the Gastrointestinal System: Anatomy, Physiology, and Assessment

SCIENTISTS IN ACTION

9.7K 次观看.  University of the Basque Country (UPV-EHU), Biocruces Health Research Institute. 在这里, 我们提出了一个协议, 以执行一个简单的细胞分离的胞质和核蛋白在人类新鲜和冰冻肠道活检。

视频: Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

科研

发育生物学

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

高速离心条件下 u937 细胞的细胞分裂

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

生物化学

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

单核RNA-seq和ATAC-seq的新鲜冷冻脑肿瘤的核隔离

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

癌症研究

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

分离细胞核用于实体瘤标本中的多组测序

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

作者聚焦：在具有挑战性的肿瘤微环境中增强用于多组测序的细胞核分离

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

生物学

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

新鲜冷冻胃肠标本的亚细胞分离

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此视频中的章节

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞

高速离心条件下 u937 细胞的细胞分裂

单核RNA-seq和ATAC-seq的新鲜冷冻脑肿瘤的核隔离

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

通过等密度梯度纯化对U937细胞进行细胞分级分离

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化