我们感谢来自博士的讨论，指导和支持。迈克尔Andreeff博士。，和Jared伯克斯博士。从MD安德森流式细胞仪和细胞成像核心设施。这项工作是由美国国家癌症研究所（RC1-CA146381，CA-083639，R01NS06994，CA116199和CA109451对于FCM部分资金。ELS是由国防部陆军部（BC083397）也支持。

同基因小鼠骨髓移植（BMT）批准的机构IACUC协议（ 注:此协议的成功需要利用任何标记的细胞类型（S）的为多光谱的分析目标，并促进下游分析，人们可以利用任何遗传或稳定的细胞标记，我们建议的任何细胞，组织或器官将要能够应用于移植模型，而移植可以包含尽可能多的独特标记的种群为研究认为是有用的。此外，多重标记的蜂窝系统，如在转基因小鼠中发现包含多个谱系限制，荧光标记的人群，可以设计，以消除移植并发症的某些病理状态的研究。 <ol><li>致死量照射骨髓移植受体小鼠的9.5戈瑞四个小时单剂量重建与捐助骨髓前。 （GFP骨髓移植受者应该是6-10周龄）。 </li><li>剪切，剥离回暴露后肢用无菌手术剪之前弄湿鼠标皮用70％乙醇。删除整个腿部，包括髂嵴在骶髂关节。通过削减略高于脚踝，然后从骨头彻底清除所有的肌肉，韧带和多余的组织丢弃的脚。冲洗骨髓从胫骨，fibia和髂嵴与使用2％无菌胎牛血清的PBS（PBS-2）一23号针头。 （BMT RFP捐助者应根据机构的标准被牺牲。） </li><li>片，并轻轻挤压剩余骨成使用手术刀和镊子的片段，然后放入50ml锥形管中与3微克/毫升胶原酶我1小时，在37℃，转速300rpm。 </li><li>顶过管子用PBS-2和收集上清到一个新的试管，并结合冲洗细胞。降速以250rpm的转速为5分钟，在4°C。 </li><li>重悬细胞于PBS-2，并通过40纳米细胞过滤器过滤。在这一点上，该细胞可以被标记为fluorescen吨激活细胞分选（FACS）如果一个人想操纵的BMT特定人群。 </li><li>重悬，每100微升PBS 2×10 6个细胞，每只小鼠。使用29号针头由尾部或眶后静脉斜角朝上系统地注入绿色荧光蛋白照射受体小鼠。注入一个鼠标用100微升PBS仅作为植入控制。用热灯前注射扩张血管和注射后用尾静脉注射克制。有无菌纱布可应用于光压到尾后注入。 </li><li>三周后BMT，收集血液眼窝并通过流式细胞术分析，以确认GFP的循环细胞的存在和不存在的RFP循环细胞。控制鼠标会在这一点上已经死亡。 </li></ol> 2。肿瘤移植植入据机构IACUC准则<ol><li>成长ID8 体内注射液（1×10 6 C前卵巢鼠的肿瘤细胞在体外每只小鼠的ELL）。注射的当天，胰蛋白酶消化细胞，用PBS洗涤，用PBS重悬在每100μl的1×10 6个细胞。 </li><li>注射肿瘤细胞到小鼠的intaperitoneal腔，针应当斜面朝上。灭菌注射的视线用70％乙醇。注入左下或腹部右下象限（颅稍内侧到下集的雌性小鼠腹部乳头），避免撞上器官。由颈背抓握住尾巴与小指或无名指抑制小鼠。小鼠可以用异氟烷麻醉用于此过程。 </li><li>牺牲小鼠时肿瘤细胞植入的迹象，比如轻微腹胀，是显而易见的。这将发生在4-12个星期。 </li><li>消费肿瘤来自IP腔。在腔内肿瘤的样子就和周围器官的表面的白色结节。用解剖刀，取出肿瘤并放置在10％福尔马林24小时固定的管/罐。组织学p赔偿可以外判予病理学/组织学的核心，如果试剂和材料不容易获得的石蜡包埋和幻灯片的准备。部组织成5-8微米切片载玻片上用于随后的染色过程。 </li></ol> 3。多参数免疫荧光染色<ol><li>准备单色控制幻灯片的每个荧光探针在实验中使用。在这种情况下，四种颜色的使用。因此，四张幻灯片为每个单独的颜色以及背景噪声的控制和完整的组合染色最后一张幻灯片一张幻灯片。 （共载玻片= 6）所有的幻灯片将被处理并排以相同的方式，以减少实验误差。 </li><li>烤石蜡切片，在56℃下反应1小时。 </li><li>洗涤载玻片3倍10分钟，二甲苯等。 </li><li>洗2×5分钟，100％乙醇中。 </li><li>洗涤1X 3分钟95％的乙醇</li><li>洗涤1X 2 min90％的乙醇。 </li><li>洗涤1X 2 min70％的乙醇。 </li><l我&gt;洗1X 2分钟水。慢慢地浸泡染色架在水中的10倍进出。 </li><li>在最后一次洗涤系列，2分钟（微波高）预煮柠檬酸钠缓冲液。 （可以用Tris EDTA缓冲液根据所使用的抗体取代） </li><li>在柠檬酸钠缓冲液（微波对10％的电力，或在高压锅）煮沸的幻灯片20分钟。如果缓冲区沸腾，关掉微波炉，让坐。 </li><li>从热源取出，放幻灯片休息30分钟，在柠檬酸钠缓冲液降温。 </li><li>冲洗载玻片在水中5分钟。 </li><li>周围的肿瘤切片用巴氏钢笔标记，并把2％BSA / 1％FBS的马来酸的封闭缓冲液1小时。单独准备的幻灯片，以不让他们在这一步干出来的。 </li><li>在封闭缓冲液中制备的初级抗体在最佳稀释。 （如果使用的抗体是所有不同种类的（或IgG链的变化，例如:IgG1的对同一物种的IgG2a的），他们可能会期间作为组合使用英格尔潜伏期。如果抗体物种重叠，然后孵育，必须依次进行，以减少抗体的交叉反应。） </li><li>在这一点上，留下两个幻灯片，只有封闭液。这两个幻灯片将作为未染色的对照和核染色控制。其他单一色彩控制将孵育个别主要抗体。只有在分析滑动将具有第一抗体的结合。 </li><li>孵育在在上缓慢旋转摇床的水分腔框幻灯片与第一抗体4℃过夜（〜16-20小时）（ 注:带4 +的荧光团，连续染色法或直接缀合工作时可能是必要的，以减少抗体的交叉反应在这情况下重复步骤1.16-1.21直到所有的抗体应用于幻灯片。） </li><li>在PBST 10分钟（2个）轻轻振动筛洗幻灯片</li><li>虽然洗是怎么回事，准备Alexafluor 488，594和647的二抗的最终稀释在封闭缓冲液中1:1000。 （确保这些抗体和稀释液保存在4℃，并在黑暗中在任何时候都保持荧光跨批次和时间的推移）。 </li><li>在单色控制幻灯片，放置单个Alexafluor稀释匹配使用的主要抗体的物种。所有的二抗将被放置在分析滑动。未染色和核深染控制幻灯片将继续留在封闭液经过该步骤。 </li><li>孵育在缓慢旋转振荡器在室温下2小时的保湿室箱（用铝箔覆盖，避免光线照射）。 </li><li> 5分钟在PBST（2个）轻轻振动筛洗幻灯片。盖上铝箔避光。 </li><li>加DAPI（1:10,000），持续1分钟到DAPI只幻灯片和分析滑动 。盖上铝箔避光。其他幻灯片可能会留​​下覆盖在洗涤容器。 </li><li>洗两DAPI孵育在第5分钟，洗涤，以不会污染其他幻灯片用DAPI染色单独的容器中滑动。对于第二个5分钟的洗涤，幻灯片可以在PBST中进行组合（所有洗涤液应在振荡器上进行的）。盖上铝箔避光。 </li><li>在水中浸简要滑梯前盖片（1.5厚度与水的安装介质保存荧光） </li><li>让干燥3小时，在黑暗中在室温。 </li><li>密封盖玻片用指甲硬化剂的边缘。 （不要使用指甲油的丙酮可淬的荧光信号）。 </li><li>立即分析幻灯片或在4℃保存，供日后分析。 </li></ol> 4。多光谱成像<ol><li>收集的OLYMPUS X-63正置显微镜与Nuance的软件，用于数据收集利用Nuance的EX摄像机（含液晶可调谐滤波器）多光谱影像。 </li><li>使用油浸物镜来提高分辨率搜集图像。 </li><li>创议l设置了包括设置光谱范围为每个使用中的荧光团。 <ol class="lalpha"><li>对于4色: <ol class="lroman"><li> DAPI:420-480nm波长</li><li> AF488:490-580nm范围内</li><li> AF594:590-640nm的</li><li> AF647:650-720nm以下</li></ol></li></ol></li><li>取分析滑动的初始图像，以获得适当的曝光时间对于每个光谱范围内的。 </li><li>创建"光谱库"，该软件将用来识别和区分彼此的信号与噪声的荧光团的比例和背景信号。 （ 注:每个荧光染料必须有自己的单一色彩控制幻灯片以组装与适当波长曲线的光谱库，以确保数据集的正确分析举例:3荧光= 4控制幻灯片; 5荧光= 6控制幻灯片）。 <ol><li>图像的未染色的幻灯片第一。这张幻灯片将频谱图书馆，A内指定Ÿ为背景 。&gt; </li><li>图像的DAPI只滑动秒。这张幻灯片将用于指定DAPI染色细胞核与"画"的工具，将谱库为DAPI内保存。&gt; </li><li>图像的AF488只滑动三分之一。这张幻灯片将用于指定AF488染色区域与"画"的工具，将谱库为AF488内保存。&gt; </li><li>图像的AF594只滑第四名。这张幻灯片将用于指定AF594染色区域与"画"的工具，将谱库为AF594内保存。&gt; </li><li>图像的AF647只滑第五名。这张幻灯片将用于指定AF647染色区域与"画"的工具，将谱库为AF647内保存。&gt; </li><li>下列各个频谱分量的集合，保存该谱库和协议。&gt; </li></ol></li><li>一旦谱库完成后，找到自己感兴趣的区域上的显微镜分析幻灯片 ，并收集/保存图像。&gt; </li></ol> 5。多光谱图像的定量分析<ol><li>导入使用的InForm软件收购Nuance的图像的代表品种。 </li><li>打开保存的谱库中所指示的程序</li><li>确定感兴趣的组织区域进行分析。 （ 如肿瘤组织与脂肪组织）。 </li><li>确定基于DAPI染色的单个细胞。细胞质将自动基于所述核的形状来限定。 </li><li>挑荧光强度读出所选择的每个荧光参数（ 如平均值，标准偏差） </li><li>一批采集的图像，并让使用该算法的软件过程。 </li><li>当图像处理结束后，合并结果文件。 </li><li>打开结果在Excel（或任何其他分析软件）和暗算对方两种荧光强度的XY轴。各荧光强度每单元给出。的双阳性细胞将出现在右上象限。用户必须定义​​基线荧光强度。 </li></ol>

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FCM，招商银行和ELS有没有冲突，并没有透露。

本文中我们描述了多光谱成像的应用中，我们描述了作为复用的细胞组合物MIMicc-多光谱询问，以分析肿瘤微环境中的源自骨髓的间质成分，但是这种方法和概念可以应用于破译了组成一个复杂的其他细胞成分组织中，例如在伤口愈合反应或再生组织中看到。在这些实验中，我们利用转基因小鼠中，以荧光从内植入肿瘤微环境中的宿主来源的细胞区分骨髓来源的细胞，但该技术适合于使用任何报告...

了解组织的发育和修复是显著，以阐明在伤口愈合，2,3再生医学，发育生物学，肿瘤生物学参与细胞成分。正在修理的情况下，许多类型的细胞渗透到周围的微环境中血管生成，细胞外基质沉积，增殖和组织结构调整提供帮助。细胞因子和表型可以基于多参数，复用标记，可以识别定位，分化状态和所研究的微环境中的细胞成分之间的相互作用来识别。在这里，我们描述肿瘤的发展作为一个典型的例子为这个多色多细胞移植模型其次是多光谱成像和光谱分离的方法。 肿瘤进展是显着的几个收购功能，包括增强的扩散，一个多步骤的过程ntiapoptotic，浸润和血管生成特性。4肿瘤的发展是由被招募到周围环境中，以提供生长因子，结构基质，血管网络和免疫调节非肿瘤细胞容易。1,5,6这种微生包括衍生自细胞的本地，邻近的组织，例如脂肪，血管和遥远来源如骨髓衍生细胞1。非赘生性细胞结合的程度依赖于来自肿瘤，这往往对应于肿瘤的阶段/级的需求。理解肿瘤微环境中支持的作用，人们必须了解的非肿瘤细胞群的起源和分化潜能。 这个协议被设计在肿瘤进展的同时通过骨髓来源的细胞组分可视化的解释，以帮助和局部组织衍生工编细胞。利用荧光报告基因表达的转基因小鼠，我们移植GFP（绿色荧光蛋白）的骨髓成致死剂量照射的RFP（红色荧光蛋白）鼠标。继成功的骨髓移植，同基因肿瘤细胞系原位注入并允许嫁接4-8个星期。将所得的肿瘤是从小鼠切下并处理用于免疫荧光（IF）染色进行可视化的基质组分。复中频标记是一种常用的技术，它涉及显著优化7-9，不过使用的是多光谱成像/解混平台提高对于具有光谱重叠的荧光标记物的组合的可能性。在此，我们提出我们称之为复用蜂窝组成MIMicc，多光谱审讯染色及分析多达内对单张幻灯片肿瘤部分8标志物，以分析细胞起源，细胞分化ST的技术ATUS和元器件的肿瘤微环境内的细胞 - 细胞相互作用。这个简化的例子有在为了分析五个，六个或更多个标记物，利用抗体或细胞内的启动子驱动的荧光表达， 表1列出潜在荧光抗体染色组合以考虑相应的物种被扩展的可能性。

<table><tbody><tr><td>.2 &amp;mu;m filter</td><td>Fisher</td><td>09-740-35A</td><td></td></tr><tr><td>10 ml lure lock </td><td>BD</td><td>309604</td><td></td></tr><tr><td>25 G needle</td><td>Cardinal</td><td>BF305122</td><td></td></tr><tr><td>40nm filter</td><td>BD</td><td>352340</td><td></td></tr><tr><td>50 ml conical tube</td><td>Fisher</td><td>1495949A</td><td></td></tr><tr><td>Alexafluor 488-ms1</td><td>invitrogen</td><td>A21121</td><td></td></tr><tr><td>Alexafluor 594-Rb</td><td>invitrogen</td><td>A21207</td><td></td></tr><tr><td>Alexafluor 647-ch</td><td>Invitrogen</td><td>A21449</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906-500G</td><td></td></tr><tr><td>Cover slips</td><td>Corning</td><td>2940-243</td><td></td></tr><tr><td>CRI-Nuance</td><td>Caliper Life Sciences</td><td></td><td></td></tr><tr><td>Dapi</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>DMEM</td><td>CellGro</td><td>10-017-CV</td><td></td></tr><tr><td>Ethanol</td><td>Dharmacon</td><td>4004-DV</td><td></td></tr><tr><td>FBS</td><td>invitrogen</td><td>16000-044</td><td></td></tr><tr><td>GFP-ms1</td><td>Abcam</td><td>ab38689</td><td></td></tr><tr><td>Insulin needle</td><td>BD</td><td>329424</td><td></td></tr><tr><td>Maleic acid</td><td>Acros</td><td>100-16-7</td><td></td></tr><tr><td>Moisture chamber box</td><td>Evergreen</td><td>240-9020-Z10</td><td></td></tr><tr><td>Mounting media</td><td>dako</td><td>S3023</td><td></td></tr><tr><td>Nail hardener</td><td>Sally Hansen</td><td>2103</td><td></td></tr><tr><td>Pap pen</td><td>Abcam</td><td>Ab2601</td><td></td></tr><tr><td>Pen/Strep L Glut</td><td>invitrogen</td><td>10378016</td><td></td></tr><tr><td>Steril PBS</td><td>invitrogen</td><td>14040-182</td><td></td></tr><tr><td>Trypsin</td><td>invitrogen</td><td>252000-56</td><td> </td></tr><tr><td colspan="4"> &lt;strong&gt;Blocking Buffer&lt;/strong&gt; &lt;table fo:keep-together.within-page=&quot;always&quot; border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;maleic acid&lt;/td&gt; &lt;td&gt;14.51 g &lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaCl&lt;/td&gt; &lt;td&gt;10.95 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaOH pellets (to 7.5pH)&lt;/td&gt; &lt;td&gt;9+g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Distilled water&lt;/td&gt; &lt;td&gt;250 ml&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt; *add 0.2% Tween-20 to 1x solution for blocking buffer with 2%BSA/2%FBS. Stir and filter.&lt;br/&gt; &lt;strong&gt;Antigen retrieval buffers:&lt;/strong&gt; &lt;table fo:keep-together.within-page=&quot;always&quot; border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Tris-EDTA Buffer &lt;br/&gt;(10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0)&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Tris Base&lt;/td&gt; &lt;td&gt;1.21 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;EDTA&lt;/td&gt; &lt;td&gt;0.37 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Distilled water&lt;/td&gt; &lt;td&gt;1000 ml (100 ml to make 10x, 50 ml to make 20x)&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Tween 20&lt;/td&gt; &lt;td&gt;0.5 ml&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt; *Store at RT for 3 months or at 4 &amp;deg; C for longer &lt;table fo:keep-together.within-page=&quot;always&quot; border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Sodium Citrate Buffer &lt;br/&gt;(10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0)&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Tri-sodium citrate (dihydrate)&lt;/td&gt; &lt;td&gt;2.94 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Distilled water&lt;/td&gt; &lt;td&gt;1000 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;HCl (adjust pH to 6.0)&lt;/td&gt; &lt;td&gt;1N&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Tween 20&lt;/td&gt; &lt;td&gt;0.5 ml&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt; </td></tr></tbody></table>

quantitative multispectral analysis following fluorescent tissue transplant for visualization of cell origins, types, and interactions

以理解的多个蜂窝单元，以一种复杂的组织的发展贡献的愿望;如参与再生组织，肿瘤形成，或血管生成中的许多细胞类型中，我们设计了在肿瘤发展的一个多彩色的细胞移植模型该细胞群的不同颜色的荧光报告基因小鼠起源和移植，嫁接或和周围一个发展中的肿瘤注射。然后，这些彩色的细胞被招募并纳入肿瘤基质。为了定量评估骨髓来源的肿瘤基质细胞，我们移植绿色荧光蛋白转基因表达全骨髓成致死剂量照射RFP批准的IA​​CUC表达的小鼠。该被原位注入移植小鼠0ovarian切除肿瘤6-8周后移植和原籍骨髓标记（GFP）以及抗体标记物来检测肿瘤相关分析基质使用多光谱成像技术。然后，我们修改我们称之为复用蜂窝组成MIMicc - 多光谱讯问的方法，使用fluoroprobes的多光谱混合像元分解定量评估这些标记的细胞来自这起群体（以原价报告基因标签），并作为我们UNMIX 4,5能力，每张幻灯片增加6个或更多频谱，我们已经添加了细胞谱系或分化增加精度相关的其他免疫组化。利用软件来检测共定位复用的荧光信号，肿瘤间质的人群可追溯，枚举和特点的基础上标记染色。1

利用多光谱成像技术来分析肿瘤植入在我们的转基因骨髓移植小鼠模型，我们能够辨别间质瘤组件是骨髓来源。初始BMT证实3周移植后通过流式细胞术（ 图1）。原位注射未标记卵巢肿瘤BMT后确认植入切除和福尔马林固定的6周后最初的肿瘤注射。石蜡包埋的肿瘤切片，切片在&lt;8μm的切片和染色。适当的单一色彩控制幻灯片的每个荧光基团包括一个用于背景自发荧光而进行。 表1?...

Watch this Scientific Journal Video about Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions at JoVE.com

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

复合组织群众，从机关到肿瘤，是由各种细胞成分的。我们阐明了利用多标记的荧光转基因小鼠与多参数免疫荧光染色结合组织随后光谱分离破译细胞起源以及基于蛋白表达细胞特征的细胞内表型的贡献。

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

quantitative-multispectral-analysis-following-fluorescent-tissue

Research

JoVE Journal

Medicine

9.3K 次观看.  MD Anderson Cancer Center. 复合组织群众，从机关到肿瘤，是由各种细胞成分的。我们阐明了利用多标记的荧光转基因小鼠与多参数免疫荧光染色结合组织随后光谱分离破译细胞起源以及基于蛋白表达细胞特征的细胞内表型的贡献。 

视频: Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

遗传学

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

生物工程

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

生物学

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

癌症研究

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

医学

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

冷冻组织部分多光谱荧光成像的快速方法

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

免疫与感染

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...