作者要感谢萨莉·罗伯茨（英国伯明翰大学，英国伯明翰）的那种的E1E4抗体的礼物。这项工作是由国家癌症研究所（4R00CA137160-03）的赠款支持。

1。器官筏文化的制备<ol><li>金属筏电网的先处理，以消除任何可能干扰与分化过程中的残留铬硫酸。一小时含硫酸的玻璃烧杯中浸泡的金属网格，然后用自来水不断冲洗过夜。筏电网隔夜冲洗后，应为3-5小时，在双蒸水冲洗。 </li><li>提供支持排上，弯曲筏约0.5厘米的距离相等，彼此的三个方面。金属网格，然后进行高压灭菌。 </li><li>准备加入2.2克NaHCO3和4.8ĞHEPES的10X重组缓冲区。溶解在100毫升0.05 M氢氧化钠。过滤消毒，并在5毫升分装保存在-20°C </li><li>准备不碳酸氢钠DMEM培养液的10倍。过滤消毒，并在5毫升分装保存在-20°C </li><li>准备小鼠表皮生长因子：溶解表皮生长因子100毫克和10毫克的BSA的每一个10毫升的去离子DDH 2 0。结合表皮生长因子和牛血清白蛋白和总容积为100毫升至80毫升的去离子DDH 2 0添加。过滤，消毒，分装（5毫升），并储存在-20°C为每公升电子介质中添加1 mg / ml的EGF（终浓度为5毫微克/毫升）5毫升。 </li></ol> 2。胶原凝胶的制备<ol><li>一个胶原蛋白凝胶是要求每个筏。胶原蛋白应保持在冰上，以防止它从巩固。每个胶原凝胶胶原蛋白组成的混合需要3毫升：2.4毫升冷鼠尾型胶原蛋白（终浓度为3-4毫克/毫升），0.3毫升的10X重组缓冲区，0.3毫升培养液10X AND1-2×10 6鼠标3T3 J2的成纤维细胞作为饲养层细胞。鼠尾胶原的高浓度的解决方案，在0.02 mol乙酸稀释，以达到正确的工作浓度。 </li><li>胶原凝胶（筏）数量确定您需要，然后计算主结构的胶原蛋白的数量，10X reconstitution缓冲区，10X DMEM和J2的成纤维细胞。由于胶原蛋白的粘度为3-4额外的凝胶在每个主结构，包括足够的。 </li><li>准备胶原蛋白凝胶，trypsinize J2的成纤维细胞和中等抵消。结合成纤维细胞和地方都在50毫升锥形瓶。计数细胞，以确定可以排上。同样，1-2X10 6成纤维细胞胶原蛋白凝胶要求每因而每筏。降速在低速的细胞。 </li><li>在的10X重组缓冲区，10X DMEM和胶原蛋白的数量计算筏适量的中等颗粒和悬浮的成纤维细胞吸。添加胶原蛋白的最后固化太快，以防止凝胶。一旦添加胶原蛋白，迅速拌匀，但轻轻地防止引入气泡的凝胶。凝胶应该是一件橘红色，指示正确的pH值。如果凝胶的混合物是太黄，添加一个过滤灭菌1N滴情侣NaOH和混合轻轻地获得正确的颜色。 </li><li>加入3毫升的胶原纤维母细胞混合井6孔培养皿。胶原蛋白的混合物，应吸管慢慢下来，每口井的一侧，以避免产生气泡。放置在37℃组织培养孵化器的板，并允许以巩固30分钟。 </li><li> 30分钟后，加入3毫升电子媒介与EGF凝固的胶原凝胶的顶部，并返回板的孵化器。凝胶应保持在37°C，至少一天，但在四天之内使用。最佳结果发生后2天的潜伏期。 </li></ol> 3。准备角质分化<ol><li>应通过角质细胞低增长约70％汇合。每个筏需要1-2X10 6角质。 Trypsinize角质和消除与E与表皮生长因子的培养基。计数细胞，以确定可以排上。降速低SPE细胞版。每个计算筏文化电子与表皮生长因子的培养基在1毫升悬浮颗粒。 </li><li>胶原凝胶吸倾斜6孔板介质。每个凝胶与EGF加入2毫升的新鲜ê介质。对于每个凝胶，小心加入1毫升的再悬浮角质电子媒介以及在已存在的2毫升。轻轻吸管角质，加2毫升的媒体，然后让秋天下拉明智放回凝胶。震动或摇晃板，可能会导致细胞分布不均的胶原蛋白凝胶的一侧。放置在37℃培养箱板。 </li><li>应细胞生长汇合，每天更换电子媒介。细胞汇合时，黄色的一天后，在介质的变化介质的变化。这通常需要2-4天。 4天如果介质没有改变，继续下一步。 </li></ol> 4。使筏文化<ol><li>用无菌镊子把无菌金属筏电网的，弯曲两侧向下，变成了10厘米的菜。吸的胶原蛋白凝胶的介质。放松以及双方的凝胶，凝胶的周长周围用无菌刮刀使用向上和向下运动。要删除的胶原蛋白凝胶，倾斜板稍微抬起放置铲下。奠定胶原蛋白凝胶的金属网格上，而不会产生气泡之间的网格和凝胶。每个格子可以放在两个凝胶。 </li><li>为了使气 - 液界面，无需添加表皮生长因子é中等慢慢盘底部，使筏电网接触媒体，但胶原蛋白凝胶是不。媒体不应该通过在网格的孔。慢慢添加介质，以避免产生气泡，根据电网，因为这将防止统一分化。在37°C孵育筏和改变介质隔日维持气 - 液界面。收获后14天的救生筏。 </li></ol> 5。代表结果当t他协议正确执行主角质，此过程将导致在分化良好的上皮细胞，在不同层次的皮肤是很明显的。实现这一目标染色苏木精伊红（H＆E）在图1B所示，筏文化节，通过组织学检查。 H＆E染色也非常有用，检查排筏，病毒或抗病毒药物效果的分层和分化能力的细胞形态。例如，从HPV-永生主的人角质形成细胞（HFK-31）的筏尤其是较厚的相比，从正常人包皮角质细胞（HFK）筏增殖率增加，反映了人乳头状瘤病毒蛋白质的能力，以维持分化细胞活跃在细胞周期（图1B）10。这个结果在保留整个上皮细胞的细胞核，而正常角质细胞分化的细胞周期退出后，在击穿核膜。更密切地审查分化，免疫组化可以进行检查特定的分化，包括角蛋白，以及外皮，聚角蛋白微丝蛋白。不同的细胞角蛋白表达在分化的特定阶段7。细胞角蛋白10（K10的），如图2所示，是不是表示在基底层，只发现在基底层层次逐步分化的细胞组成的。在HPV阳性筏，K10的表达比正常角质细胞，再次反映了人乳头状瘤病毒蛋白，角质形成细胞分化的影响，推迟。免疫组化也可以用来研究病毒基因的表达谱。图3所示是一个有代表性的染色检查的人乳头状瘤病毒蛋白素E1 ^ E4类，这是一个迟到的蛋白，其表达仅限于上皮至上层的表达。正如预期的那样，没有染色观察正常呼玛筏文化n个包皮角质。 <img alt="图1" src="/files/ftp_upload/3668/3668fig1.jpg" /> 图1）大纲的方法准备从小学角质细胞或上皮细胞株的器官筏文化。乙）苏木精和曙红染色稳定维持HPV-31（HFK-31）episomes，或从人体正常包皮角质细胞（HFK）从人类包皮角质生成的器官筏文化。确定个人的上皮细胞层，以及胶原插头。 <img alt="图2" src="/files/ftp_upload/3668/3668fig2.jpg" /> 图2角蛋白10（K10）的表达仅限于上皮的基底层层。免疫组化HFK-31细胞，以及正常使用的抗体到K10 HFKs器官筏文化的横截面。细胞DNA被用DAPI counterstained。被抓获的图像使用的conf嗓音的荧光显微镜。箭头表示上皮的基底层。 <img alt="图3" src="/files/ftp_upload/3668/3668fig3.jpg" /> 图3。人乳头状瘤病毒的E1 ^ E4类蛋白在病毒生命周期的生产阶段后期制作。免疫组化HFK-31细胞，以及正常使用抗体来E1E4 HFKs器官筏文化的横截面。细胞DNA被用DAPI counterstained。使用共聚焦荧光显微镜图像被抓获。箭头表示上皮的基底层。

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

我们什么都没有透露。

我们在这里介绍一个可以用来研究一般上皮细胞分化的方法，但也可以很容易地适应研究病毒的发病机制，以及潜在疗法的疗效。许多病毒的目标上皮细胞感染的主站点，在人乳头状瘤病毒的情况下，或在一些点在病毒的生命周期，与疱疹病毒。虽然越来越多器官筏文化是费时，忠实地在体内的上皮细胞分化概括提供了一个非常有用的方法来检查病毒：宿主细胞相互作用的能力。成功地培?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>鼠尾胶原1型（4-5mg/ml） </td><td> BD公司</td><td> 354236 </td></tr><tr><td>没有NaHC0 3 DMEM培养液</td><td> Invitrogen公司</td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td>西格玛</td><td> S5761 </td></tr><tr><td> HEPES </td><td> Calbiochem公司</td><td> 391338 </td></tr><tr><td>不锈钢金属网格</td><td>威廉姆斯和秉性有限公司</td><td> CR-03063-040-100-S的</td></tr><tr><td> Ë中等11 </td><td></td><td></td></tr><tr><td>表皮生长因子</td><td> BD公司</td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

器官的上皮筏子文化的发展提供了一个有效的研究人员在体外系统，忠实地概括上皮细胞分化。这个系统有许多用途。例如，增长角质细胞的三维器官筏文化的能力一直在研究人类乳头状瘤病毒（HPV）的1重要里程碑。人乳头状瘤病毒的生命周期是紧密相连的鳞状上皮分化2。器官的上皮筏子文化展示在这里重现整个乳头状瘤病毒的生命周期，包括病毒生产3,4,5。此外，这些竹筏文化表现不典型增生病变相似， 在体内与HPV感染后观察。因此，该系统也可用于研究上皮细胞癌，以及药物对一般的上皮细胞分化的影响。最初由Asselineau和Prunieras开发<s高达6&gt;和高班等 7修改，器官上皮筏式养殖系统已经成熟到一般，比较容易培养模型，其中包括对胶原细胞的生长，插头保持在气-液界面（图1A）。在10-14天的过程中，细胞分层和分化，形成一个完整的厚度的上皮细胞，产生特定的分化，细胞角蛋白。收获筏可组织学检查，以及标准的分子生物学和生物化学技术。在这篇文章中，我们描述了从初级人角质形成细胞的生成筏文化的方法。可以用同样的技术，建立上皮细胞株，并可以很容易地适应与利用上皮组织正常或病变的活检8。许多病毒目标或者其复制的生命周期的一部分的皮肤或粘膜上皮。在过去的几年中，使用的可行性器官筏邪教作为一个研究病毒宿主细胞相互作用的方法URES已经证明以及几个疱疹病毒，腺病毒，细小病毒，痘病毒9。因此可以适应器官筏文化研究病毒的发病机制，并测试新的抗病毒药物，这些病毒是不是永久细胞株耕地的唯一手段。

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

一个<em&gt;在体外</em&gt;方法来模仿<em&gt;在体内</em&gt;上皮细胞分化的描述。许多病毒针对病毒生命周期的一部分上皮细胞，这种方法提供了一种研究病毒的手段：宿主的相互作用，更接近于，<em&gt;在体内</em&gt;。这种技术可用于小学的角质细胞，建立细胞系，以及正常或病变的活检组织。

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

generation-organotypic-raft-cultures-from-primary-human

Research

JoVE Journal

Immunology and Infection

19.0K 次观看.  University of North Carolina-Chapel Hill. 一个在体外方法来模仿在体内上皮细胞分化的描述。许多病毒针对病毒生命周期的一部分上皮细胞，这种方法提供了一种研究病毒的手段：宿主的相互作用，更接近于，在体内。这种技术可用于小学的角质细胞，建立细胞系，以及正常或病变的活检组织。

视频: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

制备及胸腺器官切片文化中的应用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...