这项工作是由研究赠款和赠款援助农业，林业和日本渔业部的食品纳米技术项目。

1。肝脏灌注<ol><li>麻醉成年雄性大鼠（在10至15周，体重150-200克），通过腹腔注射戊巴比妥钠溶液（50毫克戊巴比妥钠/公斤体重）。 </li><li>放置在不锈钢锅的动物和70％的乙醇喷洒在其腹部和胸部。 </li><li>做一个小切断皮肤和剥离。打开一把剪刀切腹部。 </li><li>确认门静脉的位置，通过门静脉和松散丛下一个手术线程。 </li><li>丛下腔静脉和肠系膜上静脉，以防止血液回流到肝脏的远端部分。用剪刀切割膜片打开胸腔，暴露心脏。 </li><li>进入门静脉插入塑料导管（直径为2毫米），并拧紧螺纹导管周围。 </li><li>连接导管灌注系统，这是用洗涤液（钙 + 镁2 +含EGTA，0.5毫米10毫米的HEPES和碳酸氢钠 3 4.2毫米的免费的HBSS，pH值7.2）装在37预热° C。 </li><li> 开始原位灌注10毫升/分钟的速度。同时，削减在右心房壁。继续灌注10分钟。 </li><li>切换到胶原酶溶液灌注液[含10MM的HEPES和4.2mm的碳酸氢钠 3型为辅的HBSS我胶原酶（0.05％）和胰蛋白酶抑制剂（50微克/毫升），pH值7.5，灌注速度为10 - 20分钟10毫升/分钟，直到肝脏稍肿，并显示为浆膜下的肝结构解体的标志。 </li></ol> 2。脑实质丰富的肝细胞分数<ol><li>取出，仔细转移到无菌的胶原酶溶液含有25毫升的烧杯中肝脏。剪刀切碎成小块。 </li><li>添加到由此产生的细胞悬液75毫升冷的MEM。轻柔吹打后，滤液通过细胞过滤器（100微米），去除结缔组织和未消化的组织碎片。 </li><li>在50 XG在4 ° C（刹车关闭）1分钟，转移到50 ml锥形管和离心滤液。 </li><li>小心弃上清。冷的MEM重悬沉淀。 </li><li>重复2.4）的三倍。 </li></ol> 3。肝细胞混合小学文化<ol><li>暂停的DMEM含10％热灭活的FCS，补充到100微米β-巯基乙醇，10微克/毫升的胰岛素，100微克/ ml链霉素和100 U / ml青霉素，和种子组成的培养基中的细胞获得的2.5）五至十年的组织培养瓶（表面积：75厘米2）密度6.7 × 10 4细胞/平方厘米 （5 × 10 6细胞/烧瓶/ 12日-15毫升中等）。 </li><li>孵育文化flasksat 37 ° C在5％的CO 2的气氛- 95％的空气。 </li><li>更换培养基中每2至3天的时间间隔。 </li></ol> 4。巨噬细胞的选择性分离<ol><li>经过12天的文化，巨噬细胞增殖的细胞片上。通过互惠这些细胞悬浮培养瓶中摇晃，在80 - 30分钟每分钟120招在37 ° C。 </li><li>转移到100毫米的非组织文化品位塑料菜培养基。游泳池从两个T75烧瓶中期到一个塑料盘。笔芯培养瓶中生长中期和回报他们的孵化器。 </li><li>塑料菜孵育30分钟，在37 ° C在5％的CO 2的气氛- 95％的空气。巨噬细胞容易附着到非组织的文化品位，根据这个孵化过程中的塑料菜，而其他污染的细胞不。 </li><li>吸液轻轻冲洗，用PBS塑料盘。重复此步骤三次。 </li><li>加入1毫升的TrypLE Express解决方案，入菜，孵育10min后在37 ° C在5％的CO 2的气氛- 95％的空气。 </li><li>加入9毫升培养基中。所附的巨噬细胞由细胞刮刀轻轻刮去。 </li><li>转移到一个15毫升的锥形管和离心机在180 5分钟在25 ° C（刹车）XG的细胞悬液。 </li><li>弃上清，加入1毫升培养基中。大力吹打分离成单个细胞。枚举由血球计数细胞数量。 </li><li>分离步骤，可重复，在2至3天的间隔相同的文化，两个多星期的烧瓶。 </li><li>孤立的巨噬细胞可在3.1中描述的生长培养基中培养。 </li></ol> 5。代表性的成果混合成年大鼠肝细胞的原代培养的一个例子是如图2所示。实质肝细胞失去上皮细胞的形态，文化的几天内退化或转化为成纤维样细胞。然后，大约每天6至12，相位对比明亮，vigourouly一轮巨噬细胞样细胞增殖的成纤维细胞表。培养瓶中摇晃，巨噬细胞可随时暂停的培养基，并随后转移和孵化塑料菜的结果在巨噬细胞选择性粘附（图3）。早在8天可收获巨噬细胞样细胞，并数达到12至14天的最大水平。超过10 6细胞可以从T75培养瓶中多次收获在2至3天的间隔两周以上，使每个烧瓶中的细胞总数的10 7％T75培养瓶中（图3）产量。孤立的巨噬细胞的纯度分别为95％至99％，作为评估流式细胞仪或大鼠巨噬细胞特异性抗体的免疫细胞化学法，如ED - 1（图4），ED - 3，OX - 41（图4）和Iba1 9。分离出的细胞显示了积极吞噬聚苯乙烯珠（图5），增殖反应，以重组GM - CSF与LPS刺激后，9多核巨细胞形成炎症/抗炎细胞因子的分泌，如巨噬细胞的典型特征。 <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="图1" /> 图1混合原代培养大鼠肝细胞巨噬细胞样细胞的选择性分离和净化计 ​​划。 <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="图2" /> 图2原代培养大鼠肝细胞和巨噬细胞样细胞增殖。箭头表示多核巨细胞。比例尺= 100微米。从免疫学方法的杂志，Vol.360，47-55（2010）与爱思唯尔的许可9重印。 <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="图3" /> 图3。选择性分离巨噬细胞样细胞的震动和附件方法。细胞悬浮培养液中摇动烧瓶，随后转移到非组织培养级塑料菜，并在37℃孵育早在10 min后，电镀，连接盘表面，仍然暂停，而其他污染的成纤维细胞巨噬细胞样细胞。用PBS冲洗后，获得高度纯化的巨噬细胞的人口。这些细胞获得了典型的巨噬细胞的形态，经过40分钟的文化，并经常观察有丝分裂细胞（箭头）。回收的手机号码，随后从不同文化时期的烧瓶变化显示。值是一个平均± SD三至五个烧瓶。实验重复3次，数据是由不同的符号表示。比例尺= 100微米。从免疫学方法的杂志，Vol.360，47-55（2010）与爱思唯尔的许可9重印。 <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="图4" /> 图4。单克隆抗体对大鼠巨噬细胞巨噬细胞样细胞的免疫细胞化学染色。 <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="图5" /> 图5。吞噬FITC标记的巨噬细胞样细胞微球。

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

所有的动物都被保留，并在全国动物卫生研究所动物设施的经营，根据动物实验由该机构的委员会提出的指导方针和法规。

在这里，我们报告一种简单而有效的方法来获取巨噬细胞的原代培养的成年大鼠肝细胞混合。我们的方法依赖小说中的巨噬细胞的增殖活性在大鼠肝细胞混合培养，那么随后的分离和纯化这些细胞作为巨噬细胞9的生物学特性的基础上。它可能会在成人肝细胞的原代培养的混合增殖的巨噬细胞可能起源库普弗或进入实质肝细胞的一小部分污染的相关细胞。巨噬细胞，有可能造成实质肝细胞的10和其他混合的原代培养的成纤维细胞转化的具体环境变化作出回应。 总之，我们的方法提供足够数量和纯度的大鼠肝cellswithout主要使用了先进的设备和技术技能的文化巨噬细胞样细胞的隔离。此外，分离过程中可重复相同的培养瓶中，两个多星期。这种方法可能适用于其他哺乳动物物种。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>戊巴比妥钠溶液</td><td>共立Seiyaku </td><td> Somnopentyl注射液</td><td></td></tr><tr><td>汉克的平衡盐溶液（HBSS） </td><td> Invitrogen公司</td><td> 14185-052 </td><td></td></tr><tr><td>乙二醇四乙酸（EGTA） </td><td> Sigma - Aldrich公司</td><td> E - 4378 </td><td></td></tr><tr><td>胶原酶</td><td>光纯化学工业有限公司</td><td> 032-10534 </td><td>细胞解离级 （地段支票需要） </td></tr><tr><td>胰蛋白酶抑制剂</td><td> Sigma - Aldrich公司</td><td> T - 9128 </td><td></td></tr><tr><td>鹰的最低限度的基本培养基（MEM） </td><td> Sigma - Aldrich公司</td><td> M - 4655 </td><td></td></tr><tr><td>贝科的修改 鹰的培养基（DMEM） </td><td> Sigma - Aldrich公司</td><td> D - 6459 </td><td>高糖型</td></tr><tr><td>小牛血清（FCS） </td><td> Hyclone公司</td><td> SH30070.03 </td><td>热灭活56℃30分钟。 （地段支票需要） </td></tr><tr><td> TrypLE快运</td><td> Invitrogen公司</td><td> 12605 </td><td></td></tr><tr><td>贝科的磷酸盐缓冲液（PBS） </td><td> Invitrogen公司</td><td> 14190 </td><td> 钙+镁+免费2 </td></tr><tr><td> β-巯基乙醇</td><td> Sigma - Aldrich公司</td><td> M - 7522 </td><td>库存：100毫米蒸馏水</td></tr><tr><td>胰岛素</td><td> Sigma - Aldrich公司</td><td> I - 5500 </td><td>库存：10毫克/毫升0.1N盐酸</td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15070 </td><td></td></tr><tr><td>细胞过滤器</td><td> BD公司</td><td> 352360 </td><td>网眼尺寸：100微米</td></tr><tr><td>组织培养瓶</td><td>住友电木有限公司</td><td> MS - 21250 </td><td>面积：75厘米2 </td></tr><tr><td>非组织培养塑料菜</td><td> BD公司</td><td> 351005 </td><td></td></tr><tr><td>细胞刮刀</td><td>康宁公司</td><td> 3010 </td><td></td></tr><tr><td>互惠摇床</td><td>专任</td><td> NR - 1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

Kupffer细胞是肝脏特异性居民的巨噬细胞和1-3肝的生理和病理的功能发挥的重要作用。虽然肝巨噬细胞的分离方法有良好的描述4-6，大多数这些方法需要复杂的设备，如离心elutriator和技术技能。在这里，我们提供了一种新的方法来获得足够数量和纯度的示意图如图1所示，混合成年大鼠肝细胞的原代培养肝巨噬细胞。 两步灌注法7,8肝细胞分解后，大多是实质肝细胞组成的一小部分是准备和播种到T75组织的DMEM和10％FCS.Parenchymal的肝细胞组成的培养基的培养瓶中，失去上皮细胞的形态文化的几天内，退化或转化为成纤维样细胞（图2）。由于文化的收益，6天左右，相衬明亮，圆的巨噬细胞样细胞开始增殖的成纤维细胞表（图2）。巨噬细胞样细胞的增长继续达到12天左右的最高水平，覆盖烧瓶表面上的细胞片。晃动培养瓶，巨噬细胞的培养基中随时暂停。塑料菜在巨噬细胞选择性粘附的结果其后的转让和潜伏期短（图3），其他的污染细胞继续暂停。后，用PBS漂洗，附加的巨噬细胞的收获。可多次收获超过10 6细胞来自同一T75组织培养瓶中，在2至3天的间隔两周以上（图3）。孤立的巨噬细胞的纯度分别为95％至99％，流式细胞仪评估或分离的细胞免疫细胞化学大鼠巨噬细胞特异性抗体（图4）。显示后，用LPS刺激，并形成积极吞噬聚苯乙烯珠（图5），重组GM - CSF的增殖反应，炎症/抗炎细胞因子的分泌多核巨细胞9。 总之，我们提供了一种简单而有效的方法，以获得足够数量和纯度，无需复杂的设备和skills.This方法可能适用于其他哺乳动物的肝巨噬细胞。

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

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

获得原代培养大鼠肝细胞的巨噬细胞的一个新的方法来描述。这种方法利用在文化的巨噬细胞增殖，选择性附件晃动培养瓶和塑料菜的净化。这项技术有效地提供肝巨噬细胞，而无需复杂的设备和技能。

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 ...

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Immunology and Infection

37.9K 次观看.  National Institute of Agrobiological Sciences, Tsukuba, Japan. 获得原代培养大鼠肝细胞的巨噬细胞的一个新的方法来描述。这种方法利用在文化的巨噬细胞增殖，选择性附件晃动培养瓶和塑料菜的净化。这项技术有效地提供肝巨噬细胞，而无需复杂的设备和技能。

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

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 ...

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免疫与感染

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 ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

从结膜分离和表型细胞的非侵入性方式

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...