我们感谢协助设立银颗粒捕捉图像的共聚焦显微镜J. Sedgewick;部分支持由美国国立卫生研究院的研究赠款AI48484（ATH），AI20048（RA）和AI066314（CJM）;国家研究资源授予中心。 RR00169（CNPRC，美国加州大学戴维斯分校）。

使用这种方法，在研究中报道<a href="http://www.sciencemag.org/cgi/content/abstract/323/5922/1726">的李等人。，科学323，1726年至1729年（2009年</a> ） 。 结合原位四聚体染色和原位杂交（ISTH）程序 ISTH程序，可为4个步骤分为：1）抗原特异性CD8北京时间染色+ T细胞在组织; 2）原位杂交检测病毒的感染的RNA +细胞; 3）焦北京时间-染色细胞和病毒的RNA的显微成像+细胞; 4）图像分析，包括影像蒙太奇的建设和四聚体+和病毒RNA +细胞的映射。 1。北京时间染色抗原特异性CD8 + T细胞在组织切片。 <ol><li>切成200微米厚的部分使用vibratome或手术刀的新鲜组织。 </li><li>冰寒vibratome浴用无菌PBS - H和阴寒0-2 ° C。设置的vibratome叶片角度，以一个相当陡峭的27 °。只要有可能，保持组织上的冰块冷冻，以最大限度地减少退化。新鲜组织也更容易使用时，它是冷冻vibratome部分。 </li><li>切手术剪或手术刀成小约0.5厘米宽0.25厘米高件的组织。组织块，在权衡船或小盘子盖〜40 ° C融化的4％PBS缓冲融化琼脂糖低。确保组织在接触盘底部。组织块推下来，如果他们的浮动。夯实上冰。这通常需要3-5分钟。 </li><li>乐泰胶水涂层vibratome组织块。减少周围组织琼脂糖手术刀，和然后使用镊子，小心地转移到vibratome块涂上胶水脆弱的琼脂糖包埋组织。不要移动的一块组织，一旦它的组织块设置。让干燥约10分钟冰。 </li><li>摩vibratome组织块切成200um厚的部分使用死缓慢的前进速度和幅度比较高的组织。随每个vibratome速度和幅度设置。如果vibratome没有可用的，或不服从组织vibratome切片，切成细条，用手术刀组织。 </li><li>转移到中含有1毫升冷冻PBS - H的24孔组织培养板以及设置一个组织商会的画笔的部分。摆了4个组织切片到每个组织室。标签实验样品信息和安全的盖子，用橡皮筋板的组织培养板的盖子。 </li><li>另外，对于不削减与vibratome组织，如肠道，可以减少可能条状用手术刀或刀片薄。把手术刀切段，每口井只有一节。 </li><li>进入后立即完成切割组织染色。冷冻，以最大限度地减少退化，直至固定在任何时候都保持部分。不要让部分干出来的。保持在冷冻无菌PBS - H的路段。 </li><li>用0.5 mg / ml的FITC标记的四聚体的组织切片孵育过夜。可以包括鼠标或指示外抗原表位，在此孵化，如抗CD8抗体封闭液稀释1:200，非兔抗体。使用1毫升溶液，每孔和所有后续孵化，并执行和4的所有后续孵化° C板摇椅平台上。保持组织商会，含有不同的实验样品至少有一个空分开，防止交叉污染的解决方案，在后续步骤。 </li><li>初级孵化后，用冷冻PBS - H两次（2X），每次20分钟的部分。组织商会转移到不同的24孔组织培养板，含冷冻PBS - H。要小心，不滴从一个实验样品到另一个内容，当移动组织商会。对于所有后续的孵化和清洗同样组织商会转移到一个干净的盘子，包含了相应的解决方案。监察组织商会部分在程序，以确保节不卡住组织商会的双方。 </li><li>修复部分用新鲜的PBS缓冲的4％多聚甲醛在室温下2小时。洗净用冷PBS - H的2X每次5 min。 </li><li>孵育兔抗FITC抗体，如Biodesign1：10,000，阻塞为一至三天的解决方案。对于合作的标签，可以包括在细胞内的抗原表位，以及在此潜伏期的的非兔抗体。对于此选项，暴露抗原决定簇，如果需要，渗透和阻止细胞之前，第二个孵育。 </li><li>如果需要从甲醛固定细胞内的抗原表位抗原修复的使用抗体，揭露煎煮3次在0.01M的尿素，每次10秒的抗原表位，在微波炉。等待彼此之间加热2分钟。要非常小心，沸腾的解决方案，可以强制井节。如果发生这种情况，使用油漆刷推动部分从组织室的两侧或从板放回相应的组织室底部的盖子。如果不需要抗体抗原修复省略这一步。 </li><li>如果使用在细胞内的抗原决定簇的抗体，第二个孵育前，permeabolize块细胞组织切片用PBS - H的含0.3％Triton X - 100和2％正常山羊血清一小时。然后执行其余PBS - H含有0.3％的Triton X - 100和2％正常山羊血清抗体孵育。 </li><li>第二个孵育后，洗净PBS - H的部分为20分钟到几个小时。共有3个洗，重复两次。执行适当的荧光标记抗体的最终孵化。例如，羊抗兔Cy3和羊抗鼠Alexa的488在封闭液稀释1:1000每个。孵育一至三天。保持锡纸包装板块在此孵化，其后作为荧光基团的光猝灭，避光的部分。 </li><li>洗净PBS - H的部分为20分钟到几个小时。共有3个洗，重复两次。 </li><li>北京时间与原位杂交后的修复部分修复后在4％多聚甲醛为1小时到位，以确保四聚体和抗体结合，然后用PBS - H的路段再次装载。 </li><li>使用画笔转让部分载玻片。大衣与甘油/明胶含有4mg/ml N -丙基棓酸盐或其他安装媒体含有荧光团的防腐剂和盖盖玻片每节。存放在避光容器在-20℃的幻灯片的幻灯片。冲洗培养板和删除标签用酒精盖。可以重复使用板。 </li><li>共聚焦显微镜分析染色的组织切片。然后可以进行原位杂交。 </li></ol> 2。原位杂交检测感染了病毒的RNA的+细胞。 原位杂交协议，这是从先前公布的协议4,5如下修改： <ol><li>重剪5 - 8微米厚sectionss原位四聚体染色切片厚。 <ol><li> 200微米厚的部分加热到70℃，5分钟从幻灯片中分离的部分。 </li><li>一节，然后嵌入在华侨城干冰。 </li><li> 8微米的部分是厚切用低温恒温器和坚持硅烷幻灯片。 </li><li>幻灯片可以保存于-80 ° C在一个密封的盒子后续原位杂交。 </li></ol></li><li>原位杂交<ol><li>补充水分组织切片，浸泡在90％的乙醇和DEPC处理过的水，每次3分钟的幻灯片。 </li><li>通透组织切片在98 ° C水浴中加热15分钟10mm的柠檬酸缓冲液（pH值6.0）。 </li><li>酷的组织切片在室温为20-30分钟。 </li><li> DEPC处理过的水清洗两次3分钟。 </li><li>乙酰组织切片，浸泡在0.25％，在0.1 M TEA（三乙醇胺，pH8.0）醋酸酐10分钟的幻灯片。 </li><li>转移到0.1M的茶液（pH8.0）5分钟的幻灯片。 </li><li>杂交节35 S标记的特定病毒（如HIV - 1病毒，SIV）的反义探针或感（HIV - 1阴性对照，SIV）， 或35 S标记LCMV riboprobes（汇集正义和反义） 。 </li><li>洗净节两次2xSSC杂交过夜后5分钟与42 ° C。 </li><li> 5分钟洗净科技教育节（1MM EDTA，0.5 M氯化钠，20毫米的Tris - HCl，pH值7.5）。 </li><li>核糖核酸酶A和科技教育T1在37 ° C，30分钟的精华部分</li><li> 2X SSC清洗的部分，加50％甲酰胺5分钟。 </li><li>部分在1x SSC 10分钟洗净。 </li><li>在0.5 × SSC 15分钟，洗净节。 </li><li>脱水在70节，80和100％的乙醇，含0.3 M醋酸铵，每5分钟。 </li><li>核径迹乳胶外衣节</li><li>干在一个黑暗的房间1小时。 </li><li>部分暴露在4 ° C为11天（SIV）和3天（LCMV）。 </li><li>开发3分钟，在D19的部分（柯达，CAT＃146 4593），洗dH2O 30秒，修复快速定影液（柯达，CAT＃146 4106）4分钟，洗净dH2O 5分钟× 3次。 </li><li>脱水为70％，80％和无水乙醇，每5分钟。 </li><li>在荧光灯兼容的Permount介质安装幻灯片。 </li></ol></li></ol> 3。收购共聚焦显微镜图像。 <ol><li>捕获ISTH一个Bio - Rad公司MRC 1000共聚焦显微镜，或任何其他与Epi2蓝色的反射装置，激光共聚焦显微镜图像。 </li><li>使用红色的四聚体荧光，Epi2 - 522 DF32 CD8 +绿色荧光，和蓝色的反射银谷物Epi2（原位杂交信号在发达国家radioautographs）（SIV）在20X或60X Epi1 - 605（LCMV） </li><li>连续收集1微米的间隔中每个字段的三个通道的Z -序列图像。 </li><li>获得从每个部分由左到的图像根据它的行和列在Excel文件的权利，从上到下，并将其命名为每个图像。 </li><li>每幅图像捕获了〜20％，与邻国的图像重叠，以避免在重建的蒙太奇形象的差距。 </li></ol> 4。图像分析：蒙太奇图像重建，细胞的质心的计算和空间分析。 <ol><li>项目的Z -序列图像成每个通道共聚焦助理（版本4.02）。的单一形象。 </li><li>自动从三个渠道合并成一个RGB图像投射影像的，使用的Photoshop 7.0的行动过程。 </li><li>生成蒙太奇图像拼接合并后的图像在Photoshop 7.0层。 </li><li>副个别银颗粒和四聚体染色与细胞，并指定的X，Y坐标的SIV病毒RNA +和四聚体+细胞的中心（重心），使用MetaMorph（版本7.1.3）或Photoshop（作者） </li><li>导出到Excel文件作为每个单元的数字号码的质心数据。 E：T比例确定从总数的四聚体+和一节中的病毒RNA +细胞的Excel文件。 </li></ol>红四聚体+和蓝色的SIV病毒RNA之间的空间关系+细胞被捕获通过复制和粘贴到一个Photoshop文档中的Excel文件各自的地块。在减少的一个层来对齐和规模的网格，使它们重合，颜色的选择和复制和粘贴到一个新的层的蓝色的RNA +细胞的位置混浊，和然后丢弃的对齐网格层，的立场四聚体+和病毒RNA +细胞就会被夷为平地的图像显示。 5。其他注意事项如果切割传染性组织： 在BSL2.5实验室工作。采取特别的预防措施与刀片。与镊子放入vibratome刀片安装的剃须刀。 ，如果你不能把在同一个钳刀片，然后把它加入组织洗澡前，不更换刀片。当完成切割，取出刀片，一个镊子。喷雾叶片用70％乙醇或DMQ之前拆除。一如往常，处置锐器容器中的刀片。更改外层手套或消毒液冲洗干净后每个组织或受污染的PBS在坦克的联系。完成时，如果与传染性物质，添加DMQ消毒液，以PBS坦克，让我们坐一两分钟，以消除前。处理净化PBS付诸东流。然后用清水冲洗水箱，以去除盐和消毒。清洁工作区和用品DMQ。用清水冲洗用具。 为了使生物素化的MHC单体的四聚体： 获取生物素化的MHC I类分子，如HLA - A * 0201 / 2米 /或者加格（SLYNTVATL），波尔（ILKEPVHGV），或不相干的MART肽（ELAGIGILTV）贝克曼肽产生的肽分子。生物素化的HLA - A * 0301 / 2米 /肽分子要么加格（KIRLRPGGK）或NEF（QVPLRPMTYK）在NIAID的四聚体设施与生产。四聚体的生成增加6等份标记的FITC ExtraAvidin（Sigma公司），生物素马牧一个* 01 / 2米 /肽单体超过8小时，当然最后的摩尔比为4.5:1 。慢慢背后加入Extravidin的理由是，在早期的时间点有一个保证，所有的Extravidin得到所有四个生物素的网站势必过多单体。后来的反应，这个过程是效率较低，因为有少单体左，一个偶然的机会，ExtrAvidin不会有所有4个网站的约束。如果添加一次ExtrAvidin所有的反应将是效率较低，更Extravidin分子会只有2个或3个单体附加。

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The authors have nothing to disclose.

由于病毒的RNA作为病毒感染的细胞，在本报告标志物，配种前的所有试剂应RNA酶的自由。在这里描述的原位杂交程序已被修改，以保持原位四聚体染色的荧光信号。由于radioautographic原位杂交检测病毒感染的细胞信号是在一个细胞直径约的深度，原位四聚体染色所用的厚成5 - 8微米厚的部分recut。 这里描述的这种原位四聚体和原位杂交（ISTH）相结合的新技术使病毒特异性CD8 + T细胞和病毒感染的靶细胞的空间关系的同步可视化，制图和分析。这种方法可以适用于评估CD8 + T细胞为基础的疫苗和其他非病毒病原体和宿主相互作用。这里所描述的图像分析方法也可适应任何两个细胞原位交互人口研究的空间关系，例如我们最近使用过的猴免疫缺陷的非人灵长类动物模型研究HIV - 1病毒性传播的质心的映射过程病毒感染的恒河猴猕猴6

解决方案和试剂： <ul><li> PBS - H的磷酸盐缓冲生理盐水含肝素（100ug/ml或18.7U/ml）抑制核糖核酸酶</li><li>含肝素的RPMI组织培养基（100ug/ml或18.7U/ml），以抑制核糖核酸酶</li><li> 4％的PBS低熔点琼脂糖</li><li> PBS 2％正常山羊血清（或血清荧光标记的抗体作了物种）。 </li><li> PBS与2％正常山羊血清和0.3％Triton X - 100 </li><li> MHC I类四聚体结合，以FITC标记</li><li>抗FITC抗体，如生物设计兔抗FITC </li><li>荧光团标记的抗兔IgG已高度交叉使用多个标签，如羊抗兔- Cy3标记的吸附IgG抗体其他物种</li><li>荧光团标记的抗鼠IgG已高度交叉使用多个标签吸附到IgG的其他物种</li><li>的PBS缓冲的4％多聚甲醛</li><li> 0.01M尿素</li><li>甘油/明胶含有4mg/ml N -丙基没食子酸</li></ul>特种设备<ul><li> Vibratome </li><li>解剖刀</li><li>剃刀刀片</li><li>手术剪</li><li>快速设置乐泰瞬间胶（产品＃46551） </li><li>镊子</li><li> ＃2骆驼毛画笔。与刀片修剪到所需的厚度</li><li> 24孔平底组织培养板如猎鹰目录＃353226 </li><li>组织商会</li><li>锡箔</li><li>纸板幻灯片文件夹如费舍尔目录＃12-587-10 </li><li>共聚焦显微镜</li></ul>北京时间试剂快速参考 PBS - H（磷酸盐缓冲生理盐水与肝素） <ul><li> 450毫升1X PBS </li><li> 50毫升1X PBS + 10X肝素</li></ul> 1X PBS + 10X肝素<ul><li> 500毫升1X PBS </li><li> 500毫克肝素粉（干化学品仓储货架上找到） </li></ul> PBS - H / 2％，农工商超市（正常山羊血清） <ul><li> 49毫升PBS - H在猎鹰管</li><li> 1毫升农工商超市（1毫升等分-20冷冻） </li></ul>农工商PBS - H / 2％/ 0.3％Triton X - 100的的<ul><li> 49毫升PBS - H / 0.3％的Triton X - 100的猎鹰管</li><li> 1毫升农工商超市（1毫升等分-20冷冻） </li></ul> PBS - H / 0.3％的Triton X - 100 <ul><li> 500毫升PBS - H </li><li> 1.5毫升Triton X - 100的的（这是一种粘稠的液体，干化学品仓储货架上） </li></ul> 4％LMP琼脂糖<ul><li> LMP琼脂糖粉2克</li><li> 50毫升PBS </li><li>直到混合，微波炉摇匀直到粉末溶解。 </li></ul> GG - NPG（明胶与N -丙基galate甘油<ul><li>在50度水浴融化中放置一瓶甘油明胶</li><li>添加0.06克丙基galate（Terris货架上找到），摇匀</li><li>保持水浴中，偶尔摇动直到溶解。 </li><li>分装成1毫升管（棕色管） </li></ul> 4％多聚甲醛<ul><li> 4克多聚甲醛粉末（干化学品仓储货架上） </li><li>把约10毫升PBS - H的量筒中，加PF粉</li><li>添加一些水，直到量达到约70毫升</li><li>加入的NaOH的滴管。 </li><li>搅拌直至溶解（通常约20-30分钟） </li><li>加入盐酸，直到pH值达到7-8之间。 </li><li>带100毫升水的体积。 </li></ul>尿素0.01 M <ul><li>尿素0.3克（干化学品仓储货架上） </li><li>加水500毫升</li></ul>

a technique to simultaneously visualize virus-specific cd8+ t cells and virus-infected cells in situ

的数量和位置的病毒特异性CD8 + T细胞相对其感染靶细胞的数目和位置被认为是决定最终结果，范围从间隙慢性迁延性感染的关键。我们描述这里评估免疫效应（E）病毒的特异性CD8 + T细胞和（T）的感染目标，在原位四聚体（IST）的染色相结合，以检测病毒特异性CD8 + T细胞在原位之间的空间和定量关系的方法杂交（ISH）检测病毒RNA +细胞的免疫防御和感染之间的战斗发生在组织。结合IST染色和原位杂交，缩写为ISTH，使可视化的免疫效应细胞和目标，和轻便测定电子的位置和映射：T比率。反过来，这些参数可以被用来确定空间接近之间的关系，和，预测结果在早期感染的免疫反应的时间和幅度。

Watch this Scientific Journal Video about A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ at JoVE.com

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

一个技术结合原位四聚体染色原位杂交（ISTH）使的病毒特定的CD8 + T细胞其病毒感染的目标空间邻近的可视化，制图和分析，并确定这些免疫效应和目标之间的定量关系得以实现感染的结果。

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

a-technique-to-simultaneously-visualize-virus-specific-cd8-t-cells

Research

JoVE Journal

Biology

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

12.0K 次观看.  University of Minnesota. 一个技术结合原位四聚体染色原位杂交（ISTH）使的病毒特定的CD8 + T细胞其病毒感染的目标空间邻近的可视化，制图和分析，并确定这些免疫效应和目标之间的定量关系得以实现感染的结果。

视频: A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

调节性T细胞治疗移植排斥反应和I型糖尿病的治疗潜力

科研

生物学

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

归巢的骨髓造血细胞

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...