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>

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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 border="0" cellpadding="0" cellspacing="0" style="width:657px;" width="658">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">HEPES</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">H4034-1kg</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">KCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P9333-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">EDTA</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">E9884-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NaCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">5588886-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">DTT</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">10197777001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">PMSF</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">36978</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Proteinase and phosphatase inhibitors</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">A32959</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NP-40&#160;</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">CA-630</td>
			<td>Detergent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">BCA assay</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">23227</td>
			<td>Protein quantification kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Disposable plastic pestles</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">Z359947-100EA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Dounce homogeneizer</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;">431-0100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Microcentrifuge</td>
			<td style="width:128px;">Eppendorf</td>
			<td style="width:119px;">5415-R</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Shacker</td>
			<td style="width:128px;">IKA</td>
			<td style="width:119px;">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 ...

subcellular-fractionation-from-fresh-frozen-gastrointestinal

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Immunology and Infection

Unit 1

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

SCIENTISTS IN ACTION

9.6K 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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.

优化的协议<em&gt;麻风分枝杆菌</em&gt;应变管理：冷冻库存保持和维护在无胸腺裸鼠

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

从人类和小鼠心肌标本中分离巨噬细胞子集和细胞位细胞

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...