这项工作是从澳大利亚国家健康与医学研究委员会（NHMRC＃559007）和青少年糖尿病研究基金会（JDRF 4-2006-1025）和经营性基础设施计划，维多利亚州政府的拨款支持。我们感谢汤姆·曼德尔胰岛移植计划胰岛分离团队成员提供人体组织。收集和使用人体组织与当地的伦理批准（圣文森特医院HREC-A 011/04和圣文森的健康HREC-A 135/08）。

1。准备脾组织<ol><li>注意 - 所有的人力物力应被视为潜在的传染性，所有程序都必须在II级层流柜进行。用无菌剪刀和镊子，从的脾部分（约1-2厘米大小），并修剪掉尽可能多的外包膜材料尽可能消除脂肪和纤维组织。 </li><li>切一小片（1-2厘米）的脾组织，并把每一块成为一个无菌的50毫升Falcon管。 </li><li>捕捉冻结的组织浸泡在液体中N 2。 </li><li>贮存于-80℃。一个类似的协议是适用于其他组织（S）。 </li></ol> 2。人类胰岛存储准备<ol><li>文化小岛CMRL媒体。收藏报错胰岛在10毫升的锥形底部管，并通过在1,500 rpm下离心5分钟，在PBS中洗两次。倒出PBS和简要纸巾上放置的倒管排出的剩余的缓冲区。请注意，不要打跑了小岛。 </li><li>一旦倒掉，重新盖上管，液态氮和存储卡冻结在-80°C。 </li></ol> 3。准备提取物<ol><li>准备BAW（10:30:60％V / V）混合，储存于4°C。 </li><li>取下管子从-80°C。在室温下解冻。 </li><li>添加足够的冰冷的BAW覆盖片组织。小岛使用3-5毫升。对于脾组织片组织的大小取决于使用10-20毫升。 </li><li>装配的组织匀浆。由&#39;均质&#39;10-20毫升的70％乙醇/水进行清洁。 </li><li>均质化的组织在多个脉冲串，后到管放置在匀化器探针，与组织和BAW溶液。此后保持管在冰 - 铲斗。 </li><li>彻底清洁样品之间的均化器，均质化10〜20毫升的70％乙醇/水，然后BAW缓冲区。这可以防止交叉污染的组织之间samples.Dismantleand干净后，用70％乙醇/水我们E。 </li><li>是必需的，如果仅含有水溶性材料的提取物离心匀浆组织提取物在RT下在4,000 rpm，10分钟。如果更粗提物是必需的，在RT下以1,000 rpm旋转5分钟。用于提取的BAW不溶性蛋白质的最合适的方法取决于下游分析的蛋白质。对于使用的功能的免疫学测定方法中，我们建议尝试在8M脲溶解不溶性成分，因为我们先前已经发现这是很好的耐受性6。 </li><li>上清转移到一个干净的试管，并把它放在冰。 </li><li>确定使用的BCA测定法或类似的提取物中的蛋白质浓度。 </li></ol> 4。冷冻干燥提取物<ol><li>根据蛋白质所需的质量（ 即 100微克每管），相应的稀释匀浆，并免除取到标有"5.0毫升无菌，猎鹰（12x75毫米）管。我们经常使用的100微克/管。 </li><li>使用一个18-20号无菌注射器针头，使每个管帽的3个孔。 </li><li>或者通过将它们放置〜10分钟，或&gt; 1小时，在-80℃的冰箱中，在干冰上冷冻管。储存在-80°C至准备把冻干机。 </li><li>切换冷冻干燥器上，并允许平衡（-100℃）。这需要大约30分钟。 </li><li>视音量而定，冷冻干燥，可以在3小时内完成，但我们经常离开我们的样本中过夜。 </li><li>在干燥循环完成后，关闭真空泵和慢慢允许进入腔室的压力。卸下机架上。 </li><li>在无菌罩，取出穿孔的上限从管，并更换新的（猎鹰352032）。 </li><li>于-20℃下存储管样品可重新溶解在培养基中，或其他缓冲液，用于在功能上或生化分析。 </li></ol> 5。代表性的成果 图1示出的一种蛋白质的染色凝胶装载，与脾脏提取物和胰岛耗尽胰腺组织（腺泡）和纯化的人胰岛（标记小岛）。结果显示，不同分子量的蛋白质为每一个组织良好的代表性。 的组织提取物刺激人类T-细胞增殖的能力，使用的CFSE基于增殖试验7（ 图2）进行了测试。在该测定中使用的PBMC中分离出从个人与1型糖尿病。 CFSE 昏暗每5,000 CD4 +，CFSE明亮的细胞没有细胞抗原的数量的比例的幅度响应表示为:CFSE 昏暗的细胞每5000 CD4 +，CFSE明亮的细胞与抗原一式三份样品7。结果表明弱，但检测，增殖腺泡（CD1 = 3.5）和更强的反应胰岛提取物（6.8）。作为流感病毒灭活（CDI = 142.6）阳性对照。 <img alt="图1" src="/files/ftp_upload/4230/4230fig1.jpg" /> 图1。蛋白质凝胶。 <img alt="图2" src="/files/ftp_upload/4230/4230fig2.jpg" /> 图2。从CFSE增殖实验对腺泡细胞和胰岛提取物的结果。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">点击此处查看大图</a> 。

<ol>
	<li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+measuring+human+islet-antigen+specific+T+cell+function+in+type+1+diabetes.">Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.</a> Clin. Exp. Immunol. 162, 197-209 (2010).</li><li>Beckhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+T+cell-based+identification+of+human+tumor+tissue+antigens+by+automated+two-dimensional+protein+fractionation.">Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.</a> J. Clin. Invest. 120, 2230-2242 (2010).</li><li>Mannering, S. I., Brodnicki, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+CD4++T-cell+specificity+and+function+in+Type+1+diabetes.">Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes.</a> Expert Review of Clinical Immunology. 3, 557-564 (2007).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-chain+of+insulin+is+a+hot-spot+for+CD4++T+cell+epitopes+in+human+type+1+diabetes.">The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.</a> Clin. Exp. Immunol. 156, 226-231 (2009).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+A-chain+epitope+recognized+by+human+T+cells+is+posttranslationally+modified.">The insulin A-chain epitope recognized by human T cells is posttranslationally modified.</a> J. Exp. Med. 202, 1191-1197 (2005).</li><li>Moon, H. C., Joffe, M., Thomas, H. E., Kay, T. W. H., Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+extracting+tissue+proteins+for+use+in+lymphocyte+function+assays.">A method for extracting tissue proteins for use in lymphocyte function assays.</a> Journal of Immunological Methods. 359, 56-60 (2010).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J. Immunol. Methods. 283, 173-183 (2003).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cell+Proliferation+in+Response+to+GAD+and+Proinsulin+in+Healthy,+Pre-diabetic,+and+Diabetic+Donors.">CD4+ T Cell Proliferation in Response to GAD and Proinsulin in Healthy, Pre-diabetic, and Diabetic Donors.</a> Ann. N.Y. Acad. Sci. 1037, 16-21 (2004).</li><li>Mannering, S. I., Purcell, A. W., Honeyman, M. C., McCluskey, J., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-cells+recognise+N-terminally+Fmoc-modified+peptide.">Human T-cells recognise N-terminally Fmoc-modified peptide.</a> Vaccine. 21, 3638-3646 (2003).</li><li>Peakman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+preparations+of+GAD65,+proinsulin,+and+the+islet+tyrosine+phosphatase+IA-2+for+use+in+detection+of+autoreactive+T-cells+in+type+1+diabetes:+report+of+phase+II+of+the+Second+International+Immunology+of+Diabetes+Society+Workshop+for+Standardization+of+T-cell+assays+in+type+1+diabetes.">Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.</a> Diabetes. 50, 1749-1754 (2001).</li><li>Honey, K., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+cysteine+proteases+regulate+antigen+presentation.">Lysosomal cysteine proteases regulate antigen presentation.</a> Nat. Rev. Immunol. 3, 472-482 (2003).</li></ol>

没有利益冲突的声明。

此协议是因为我们想生成人体组织是免费的，如清洁剂有毒化学品的提取物。具体来说，我们已经用它来 ​​制备人体组织的提取物，可用于检测在体外人体免疫功能。使用该协议制备的提取物可以同样在任何缓冲器重新配制，并用于许多生化分析，如蛋白质印迹法或液相色谱法。这使得该技术适用于许多下游应用。 使用我们的协议中，组织提取物的反应并不强烈。?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 5毫升12×75毫米无菌聚苯乙烯管</td><td> BD猎鹰</td><td> 352054 </td><td></td></tr><tr><td>管聚苯乙烯管上限（上图） </td><td> BD猎鹰</td><td> 352032 </td><td></td></tr><tr><td> 50毫升无菌试管</td><td> Becton Dickinson公司</td><td> 352070 </td><td></td></tr><tr><td>乙腈</td><td> Mallinckradt化学品</td><td> 2856-10 </td><td></td></tr><tr><td>丁-1 - 醇</td><td> Sigma Aldrich公司</td><td> 537993-IL </td><td></td></tr><tr><td>均质:PRO200 </td><td>生物战略</td><td> 01-01200 </td><td> 10 x 115毫米的锯齿发生器</td></tr><tr><td>冻干机</td><td></td><td></td><td> VIRTIS，台式4K </td></tr><tr><td>无菌针头18-20计</td><td> Becton Dickinson公司</td><td> REF 302032 </td><td></td></tr><tr><td> CMRL-1066中</td><td>西格玛</td><td> C0422 </td><td></td></tr><tr><td> PBS </td><td>西格玛</td><td> D8537 </td><td></td></tr></tbody></table> 表1中。具体的试剂和设备。

extraction of tissue antigens for functional assays

许多的适应性免疫反应的抗原的目标，已确认由B细胞和T细胞，还没有被定义1。这是特别真实的，在自身免疫性疾病和癌症的2。我们的目的是研究人类T细胞识别的抗原的自身免疫性疾病，1型糖尿病患者1,3,4,5。来分析人体T-细胞的反应，对其中没有确定T细胞所识别的抗原的组织中，我们开发了一种方法来提取蛋白质抗原从人类组织中的格式是兼容的功能分析6。无法测量，因为在此之前，T细胞的反应，未纯化的组织提取物的提取方法产生的裂解物，所含的清洁剂对人体外周血单个核细胞是有毒的。在这里，我们描述一个协议，用于人类T细胞没有毒性的格式是从人体组织中提取蛋白质。该组织均化的混合物中的丁-1 - 醇，乙腈和笏ER（BAW）。的组织提取物中的蛋白质浓度的测定，和公知的蛋白质的质量分装到管。提取后，将有机溶剂，通过冷冻干燥除去。冻干组织提取物被保存，直到需要。用于检测免疫功能，免疫细胞的悬浮液中，在适当的培养介质中，可以直接添加到冻干提取物。外周血单个核细胞的细胞因子的产生及细胞增殖，在响应于用此方法制备的提取物，很容易测定。因此，我们的方法可以作为一个源的抗原的T细胞应答的分析，可用于人体组织裂解物的迅速地制备。我们认为，这种方法将有助于分析适应性免疫反应的组织移植，癌症和自身免疫。

Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Extraction of Tissue Antigens for Functional Assays

一个简单的协议，用于制备的人体组织的提取物被用作功能性T细胞分析中的源的抗原进行说明。此方法允许要被测量的组织来源的抗原的T细胞的反应，以<em在体外</em&gt;。

提取组织抗原的功能分析

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

extraction-of-tissue-antigens-for-functional-assays

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.8K 次观看.  St. Vincent's Institute of Medical Research. 一个简单的协议，用于制备的人体组织的提取物被用作功能性T细胞分析中的源的抗原进行说明。此方法允许要被测量的组织来源的抗原的T细胞的反应，以

视频: Extraction of Tissue Antigens for Functional Assays

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

科研

生物化学

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

神经科学

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

免疫与感染

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

医学

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

生物工程

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

小鼠肺中抗原特异性 T 细胞的分离和表征

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

作者聚焦:使用四聚体检测小鼠肺中抗原特异性 T 细胞的优化方案

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...

提取组织抗原的功能分析

摘要

探索更多视频

此视频中的章节

个性化肽阵列在器官移植 HLA 抗体检测中的研究

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

检测和罕见富集抗原特异性B细胞的表型和功能分析

与人体组织转化为癌症研究工作

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

从原发肿瘤的免疫细胞的分离

小规模的核提取物，基因表达机具功能分析

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞