Name: preparation of cytoplasmic and nuclear long rnas from primary and cultured cells
Uploaded: 2023-04-07T13:20:00
Description: Watch this Scientific Journal Video about Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells at JoVE.com

由美国血液学会、罗伯特·伍德·约翰逊基金会、多丽丝·杜克慈善基金会、爱德华·埃文斯基金会和国家癌症研究所（1K08CA230319）资助。

K562细胞用于本研究。该协议已经过优化，适用于1 x 106-5 x 10600 万个细胞。然而，通过适当增加体积，该程序可以扩大到更大的细胞量。
1.0.25x裂解缓冲液的制备
<ol><li>通过混合 表1A中提供的以下组分制备0.25x裂解缓冲液。 注意：样品需要大约 300 μL 的 0.25x 裂解缓冲液。推荐体积 = 样品数量 x 300 μL。</li>
</ol>2...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+localization:+message+on+the+move.">mRNA localization: message on the move.</a> Nature Reviews Molecular Cell Biology. 2 (4), 247-256 (2001).</li><li>Carlevaro-Fita, J., Johnson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+positioning+system:+Understanding+long+noncoding+RNAs+through+subcellular+localization.">Global positioning system: Understanding long noncoding RNAs through subcellular localization.</a> Molecular Cell. 73 (5), 869-883 (2019).</li><li>Voit, E. O., Martens, H. A., Omholt, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=150+years+of+the+mass+action+law.">150 years of the mass action law.</a> PLOS Computational Biology. 11 (1), 1004012 (2015).</li><li>El-Tanani, M., Dakir el, H., Raynor, B., Morgan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+nuclear+export+in+cancer+and+resistance+to+chemotherapy.">Mechanisms of nuclear export in cancer and resistance to chemotherapy.</a> Cancers (Basel). 8 (3), 35 (2016).</li><li>Turner, J. G., Dawson, J., Sullivan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+export+of+proteins+and+drug+resistance+in+cancer).">Nuclear export of proteins and drug resistance in cancer).</a> Biochemical Pharmacology. 83 (8), 1021-1032 (2012).</li><li>Boesenberg-Smith, K. A., Pessarakli, M. M., Wolk, D. 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在整个方案中，采取了一些步骤来优化元件，使其对感兴趣的细胞系最有效。虽然协议中的步骤相对简单，但在协议的关键方面可能需要分析和细微调整。方案中最关键的步骤是根据感兴趣的细胞系和细胞靶标将裂解缓冲液的浓度修改为适当的浓度。由于裂解缓冲液的使用在很大程度上取决于细胞膜的破坏，因此裂解缓冲液在不同细胞系上的有效性存在自然差异，因为不同细胞系之间的细胞脆性?...

细胞分离成亚细胞成分允许分离和研究定义的生化结构域，并有助于确定特定细胞过程的定位以及这可能如何影响其功能1。从不同的细胞内位置分离RNA可以提高转录水平事件以及细胞核和细胞质之间其他相互作用的遗传和生化分析的准确性，这是当前方案2的主要目的。该协议的开发旨在确保细胞质和核RNA的分离，以确定它们在核输出中各自的作用，并了解细胞核和细胞质中RNA的亚细胞定位如何影响其在细胞过程中的功能。使用来自典型实验室环境的材料，可以在不损害结果质量的情况下比以前建立的方案更有效、更便宜地实现细胞核和细胞质分离3。
此外，可以通过分离这些区域直接研究细胞质和细胞核之间的分子交换。更具体地说，了解转录组对于理解发育和疾病至关重要。然而，RNA在任何给定时间可能处于不同的成熟水平，并且可能使下游分析复杂化。该协议允许从核和细胞质亚细胞组分中分离RNA的能力，这可以帮助RNA的研究，并允许更好地了解感兴趣的特定RNA，例如非编码RNA的定位或细胞核内剪接连接的分析。
由于所使用的RNA纯化柱的大小选择性，该协议已针对分离细胞质和核长RNA（包括mRNA，rRNA和长非编码RNA（lncRNA）进行了优化，可以对其进行修饰以分离其他感兴趣的RNA。以前，长RNA的功能，如mRNA和lncRNA，高度依赖于它们各自在细胞内的定位4，5。因此，从细胞核到亚细胞结构域的输出研究变得更加有针对性的是了解输出RNA或其他细胞成分对细胞的作用。lncRNA就是一个典型的例子，因为它们的翻译和后续效应在很大程度上依赖于与其他形式的RNA6的接近和相互作用。此外，细胞核和细胞质区域之间的细胞元件交换与对各种癌症治疗的耐药机制有关7。细胞内区室的分离允许核输出抑制剂的发展，这减轻了对不同疗法的耐药机制的影响8。
分离核和细胞质RNA后，执行步骤以纯化感兴趣的RNA。由于RNA纯化试剂盒通常在实验室中发现，并且具有纯化和分离长RNA的功能，因此它们可以很好地满足本协议的目的。对于RNA纯化，产生大于1.8的260：280比率对于确保RNA测序或其他需要高纯度和分离的类似程序的样品质量至关重要。不规则的 260：280 值表示苯酚污染，表明分离效果差，结果不准确9.
一旦RNA纯化完成并且确认260：280值高于可接受的范围，则使用qPCR验证核和细胞质级分的分离结果。在此过程中，使用特定于感兴趣区域的引物来证明核和细胞质分离水平。在该协议中，MALAT1和TUG1分别用作核和细胞质标志物10。总之，它们允许使用MALAT1证明高水平的核分离，而细胞质分离预计较低。相反，当使用TUG1时，预计细胞质分级水平将高于核分离水平。
利用该协议，可以根据RNA在细胞中的作用位置分离RNA。由于在该实验中广泛使用许多材料，并且仅使用裂解缓冲液和基于密度的离心技术，因此对其他RNA类型和其他细胞组分的适用性很普遍。这可以通过阐明特定于位置的表达事件来提供重要信息，否则如果不进行分离，这些事件将无法区分。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent Tapestation</td>
			<td>Agilent</td>
			<td>G2991BA</td>
			<td>The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples.&#160;</td>
		</tr>
		<tr>
			<td>0.5% Nonidet P-40</td>
			<td>Thermo-Fischer</td>
			<td>28324</td>
			<td>Used in the making of lysis buffer</td>
		</tr>
		<tr>
			<td>50 mM Tris-Cl pH 8.0</td>
			<td>Thermo-Fischer</td>
			<td>15568025</td>
			<td>Used in the making of lysis buffer</td>
		</tr>
		<tr>
			<td>MALAT1 GE Assay</td>
			<td>Thermo-Fischer</td>
			<td>Hs00273907_s1</td>
			<td>Utilized for confirmation of Nuclear fraction.</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-Well Reaction Plate with Barcode</td>
			<td>Thermo-Fischer</td>
			<td>4326659</td>
			<td>The Applied Biosystems MicroAmp Optical 96-Well Reaction Plate with Barcode is optimized to provide unmatched temperature accuracy and uniformity for fast, efficient PCR amplification. This plate, constructed from a single rigid piece of polypropylene in a 96-well format, is compatible with Applied Biosystems 96-Well Real-Time PCR systems and thermal cyclers.</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Thermo-Fischer</td>
			<td>4311971</td>
			<td>The Applied Biosystems MicroAmp Optical Adhesive Film reduces the chance of well-to-well contamination and sample evaporation when applied to a microplate during qPCR</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-023</td>
			<td>Phosphate-buffered saline (PBS) is a balanced salt solution that is used for a variety of cell culture applications, such as washing cells before dissociation, transporting cells or tissue samples, diluting cells for counting, and preparing reagents.</td>
		</tr>
		<tr>
			<td>Qiagen RNA Clean Up Kit-Rneasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>RNA cleanup kits enable efficient RNA cleanup of enzymatic reactions and cleanup of RNA purified by different methods. Includes RW1, RLT, RW1 buffers mentioned throughout protocol.</td>
		</tr>
		<tr>
			<td>QuantStudio 6</td>
			<td>Thermo-Fischer</td>
			<td>A43180</td>
			<td>qPCR software utilized during protocol. Includes Design and Analysis Software for analyzing fractionation samples</td>
		</tr>
		<tr>
			<td>RLT Buffer</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>Lysis Buffer from RNA clean-up kit</td>
		</tr>
		<tr>
			<td>RPE Buffer</td>
			<td>Qiagen</td>
			<td>1018013</td>
			<td>Wash Buffer from RNA clean-up kit</td>
		</tr>
		<tr>
			<td>RW1 Buffer</td>
			<td>Qiagen</td>
			<td>1053394</td>
			<td>Wash Buffer from RNA clean-up kit</td>
		</tr>
		<tr>
			<td>Taqman Gene Expression Assays</td>
			<td>Thermo-Fischer</td>
			<td>4331182</td>
			<td>Applied Biosystems TaqMan Gene Expression Assays represent the largest collection of predesigned assays in the industry with over 2.8 million assays across 32 eukaryotic species and numerous microbes. TaqMan Gene Expression assays enable you to get results fast with no time wasted optimizing SYBR Green primers and no extra time spent running and analyzing melt curves.</td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Thermo-Fischer</td>
			<td>4305719</td>
			<td>TaqMan Universal PCR Master Mix is the ideal reagent solution when you need a master mix for multiple 5&#39; nuclease DNA applications. Applied Biosystems reagents have been validated with TaqMan assays and Applied Biosystems real-time systems to ensure sensitive, accurate, and reliable performance every time.</td>
		</tr>
		<tr>
			<td>TUG1 GE Assay</td>
			<td>Thermo-Fischer</td>
			<td>Hs00215501_m1</td>
			<td>Utilized for confirmation of Cytoplasmic fraction.</td>
		</tr>
		<tr>
			<td>UltraPure DEPC-Treated Water</td>
			<td>Thermo-Fischer</td>
			<td>750024</td>
			<td>UltraPure DEPC-treated Water is suitable for use with RNA. It is prepared by incubating with 0.1% diethylpyrocarbonate (DEPC), and is then autoclaved to remove the DEPC. Sterile filtered.</td>
		</tr>
	</tbody>
</table>

preparation of cytoplasmic and nuclear long rnas from primary and cultured cells

多年来，细胞内成分的分离一直是细胞生物学的关键工具，并且已经能够提供有关其位置如何影响其功能的有用见解。特别是，核和细胞质RNA的分离在癌细胞和寻找药物新靶点的背景下变得很重要。当许多所需材料可以在典型的实验室环境中找到时，购买用于核细胞质RNA提取的试剂盒可能很昂贵。使用本方法可以替代更昂贵的试剂盒或其他耗时的过程，只需自制的裂解缓冲液、台式离心机和RNA分离纯化柱即可分离核和细胞质RNA。裂解缓冲液用于温和地裂解细胞的外膜，而不会影响核包膜的完整性，从而释放其细胞内成分。然后，可以通过简单的离心步骤分离细胞核，因为它们具有比裂解溶液更高的密度。离心用于根据其密度差异分离这些区域，以分离细胞核中的亚细胞元素和细胞质中的亚细胞元素。离心分离出不同组分后，使用RNA净化试剂盒纯化RNA含量，并进行qPCR以验证分离质量，通过不同级分中核和细胞质RNA的量进行定量。实现了统计学上显着的分离水平，说明了该方案的有效性。此外，该系统可以适用于分离不同类型的RNA（总RNA，小RNA等），从而可以有针对性地研究细胞质 - 细胞核相互作用，并有助于了解驻留在细胞核和细胞质中的RNA功能的差异。

为了确保实现核和细胞质分离，进行了qPCR以验证结果。在此过程中，使用特定于感兴趣区域的引物来证明核和细胞质分离水平。在这项研究中，MALAT1和TUG1分别用作核和细胞质引物。总之，它们允许证明高水平的核分级分离，MALAT1作为核元素的阳性对照，而细胞质分离预计较低。相反，当TUG1作为细胞质元件的阳性对照存在时，预计细胞质分离水平将高于核分离水平。这可以在 图2</...

Watch this Scientific Journal Video about Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells at JoVE.com

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells

本协议提供了一种高效灵活的方法，使用培养的细胞从核和细胞质级分中分离RNA，然后使用qPCR进行验证。这有效地替代了其他RNA制备试剂盒。

从原代细胞和培养细胞制备细胞质和核长RNA

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how ...

该协议对于评估细胞核和细胞质中定义的生化元件的不同作用以及分析各种细胞系内这些结构域之间的细胞内相互作用是必要的。该技术利用常见的实验室设备和试剂更有效地分离细胞质和细胞核部分。该协议可以比其他协议更便宜，更快。随着对细胞质和核相互作用的研究越来越广泛，定位对细胞内事件的影响可以得到更好的理解。可以分析细胞质和细胞核之间的交换，表明这些交换过程中的某些分子是否会导致癌症的发展，正如对核蛋白的研究表明的那样。方案中最关键的步骤是将裂解缓冲液的浓度修改为适当的浓度。由于裂解缓冲液的使用在很大程度上取决于细胞膜的破坏，并且裂解缓冲液在不同细胞系上的有效性存在天然差异。首先，使用1.5毫升管在2000G下离心5分钟沉淀细胞，使用吸液管丢弃上清液。将沉淀重悬于300微升冰冷的裂解缓冲液中。然后，在 12， 000 摄氏度下以大约 4 摄氏度的速度旋转试管两分钟。小心地除去上清液并将其放入新的1.5毫升管中。剩余的颗粒将是核部分。接下来，向沉淀中加入 500 微升冰冷的 PBS，并在室温下以 2000 G 离心五分钟。为了确认使用qPCR分离区室，首先，使用市售试剂盒将RNA转化为互补DNA。准备逆转录酶预混液。添加模板 RNA。在热循环仪中孵育反应。继续进行定量聚合酶链反应和分析。首先用DEPC水稀释cDNA合成，最终浓度为每毫升20纳克。使用PCR预混液，使用手动说明制备每个样品的反应物。获取检测细胞质和细胞核分离所需的商业探针。使用MALAT1作为核标志物，TUG1作为细胞质标志物。通过将单个样品的每个技术重复的每个组分乘以 3 来计算每个组分的体积。对于每个核和细胞质样品，用MALAT1获取核级分，用MALAT1获取细胞质级分，用TUG1获取核级分，用TUG1获取细胞质级分。短暂涡旋以混合溶液，并将20微升混合物转移到光学反应板的每个孔中。用用于qPCR的透明粘合膜覆盖板，并短暂离心板以消除气泡，然后在室温下以300倍G旋转样品5分钟。使用设计和分析软件，为循环参数选择标准曲线。使用 qPCR 软件，选择安装程序运行。在数据文件属性页上，选择方法和输入周期参数。输入循环参数后，选择板片并输入要运行的样品。选择“开始运行”。运行完成后，使用样品名称、目标名称和定量周期创建数据电子表格。从MALAT1样品开始计算核分数。从马拉特1核部分减去MALAT1细胞质部分。继续使用MALAT1样品以计算细胞质部分。从 MALAT1 细胞质部分中减去 MALAT1 核部分，然后计算两个升高到负 delta CQ 的分数。使用 TUG1 样本执行计算。当TUG1作为细胞质元件的阳性对照存在时，预计细胞质分级水平将高于核分离水平。阴性结果，其中用MALAT1在样品中观察到细胞质分级分离水平。这表明从核级分中分离的RNA受到污染，可能是由于裂解浓度过低或过高。MALAT1和TUG1与细胞核和细胞质分离的相关性最高，通过蛋白质印迹和RNA电泳证实，证实了样品的纯度和质量。此外，核提取物和细胞质提取物的纯度可以通过蛋白质印迹来证明，蛋白质印迹使用层粘连蛋白作为核级分的标记物，α-微管蛋白作为细胞质部分的阳性标记物。在细胞质RNA电泳中未观察到峰，表明细胞质RNA样品中质量高，污染很少或没有。裂解缓冲液的使用对于适当的分级分离至关重要，将裂解缓冲液浓度优化为所需的目标细胞系至关重要。该技术允许对核和细胞质相互作用进行有针对性的研究，这广泛适用于各种研究主题，并允许更好地了解特定生物元素的定位如何与其功能相关。

preparation-cytoplasmic-nuclear-long-rnas-from-primary-cultured

Research

JoVE Journal

Cancer Research

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

科研

癌症研究

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

矫形子宫内膜癌模型，具有从 Vivo 传播和培养的 VX2 细胞中制成的再生淋巴病

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

毛细管电泳质谱法提取培养细胞中的水代谢物进行代谢组学分析

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the mechanical properties of the cells is often challenging. Conventional measurement methods based on contact such as compression or stretching are prone to cause cell damage, affecting measurement accuracy and subsequent cell culture. Measurements in adherent state can also affect accuracy, especially after irradiation since ionizing radiation will flatten cells and enhance adhesion. Here, a cell mechanics measurement system based on acoustofluidic method has been developed. The cell compressibility can be obtained by recording the cell motion trajectory under the action of the acoustic force, which can realize fast and non-destructive measurement in suspended state. This paper reports in detail the protocols for chip design, sample preparation, trajectory recording, parameter extraction and analysis. The compressibility of different types of tumor cells was measured based on this method. Measurement of the compressibility of nucleus was also achieved by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. Combined with the molecular level verification of immunofluorescence experiments, the cell compressibility before and after drug-induced epithelial to mesenchymal transition (EMT) were compared. Further, the change of cell compressibility after X-ray irradiation with different doses was revealed. The cell mechanics measurement method proposed in this paper is universal and flexible and has broad application prospects in scientific research and clinical practice.

Here a protocol is presented to build a fast and non-destructive system for measuring cell or nucleus compressibility based on acoustofluidic microdevice. Changes in mechanical properties of tumor cells after epithelial-mesenchymal transition or ionizing radiation were investigated, demonstrating the application prospect of this method in scientific research and clinical practice.

基于声流体微器件的细胞和细胞核可压缩性测量

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the ...

来自新鲜组织的胰腺癌来源的肿瘤类器官和成纤维细胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

作者聚焦：从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...