我们感谢脂肪组织的捐赠者和研究人员, 他们收集并提供这一资源给我们进行研究使用。我们想确认 Allergan 的资金支持。我们还感谢奥克兰大学, 医学和健康科学学院以绩效为基础的研究基金, 用于资助录像和编辑的费用, 以便提交本条。

1. 为鉴别化验准备陶瓷
<ol><li>在无菌组织培养罩中制备组织培养试剂和处理活细胞。</li>
	<li>制备 A0 培养基: Dulbecco 改性鹰中、火腿 F12 培养基 (DMEM/F12)、1x glutamax、1x 青霉素/链霉素。</li>
	<li>准备完整的 ASC 培养基: DMEM/F12, 1x glutamax, 1x 青霉素/链霉素, 10% 胎牛血清。</li>
	<li>使用粘附纯化陶瓷, 在 T75 培养瓶中展开。使用荧光活化细胞分类 (陶瓷)10确认纯度。</li>
	<li>分离细胞通过从 T75 培养瓶中去除所有培养基并加入2毫升细胞离解酶。</li>
	<li>将培养瓶留在孵化器 (37 °c, 加湿, 5% CO2) 为5-10 分钟。把培养瓶从孵化器中取出, 并牢牢地敲打瓶子的侧面。用倒置显微镜检查细胞是否与培养瓶分离。</li>
	<li>在 T75 培养瓶中加入13毫升的 A0 培养基。</li>
	<li>将15毫升转化为15毫升离心管和离心机, 在 400 x g 处为5分钟。</li>
	<li>放弃上清, 并在1毫升的完全 ASC 培养基中重新悬浮细胞颗粒。</li>
	<li>使用 hemocytometer 执行单元格计数。</li>
	<li>在完整的 ASC 培养基中稀释细胞悬浮液, 使其浓度为2.5万细胞毫升-1 。</li>
	<li>将 A0 介质的200µL 添加到96井板 (平底) 的所有外围井中。这降低了含有中心细胞的井的蒸发速率。</li>
	<li>将200µL 的细胞悬浮液转移到 microwell 板上, 以达到每井 5 x 103单元的最终细胞密度。每个治疗组需要四口水井 (三项技术重复和免疫细胞化学 (ICC) 的无主抗体控制)。</li>
	<li>离开 microwell 板在孵化器 (37 °c, 加湿, 5% CO2), 直到细胞达到汇合 (3-4 天)。</li>
</ol>2. 诱导分化陶瓷
<ol><li>用1µM dexamethasome、10µM 胰岛素和200µM 吲哚美辛补充完整的 ASC 培养基, 制备脂肪分化培养基。完全分化培养基稳定在4°c 1 月, 所以只做1的化验要求。存储在4°c 和当需要加热整除到37°c 在水浴。</li>
	<li>3-4 天后, 将 microwell 板从孵化器中取出, 用脂肪分化培养基取代一半培养基。</li>
	<li>每2-3 天执行一次中等变化。 注: 最佳脂肪分化需要14天的培养在分化培养基。用传统染料染色和基因特异污渍分析分化细胞。</li>
</ol>3. 鉴别染色-传统染料基污渍
<ol><li>用 PBS 稀释16% 甲醛, 制备4% 甲醛溶液。仅稀释实验所需的用量, 并在离心管中密封牢固。 注意: 甲醛是一种潜在的致癌物质, 在吸入时会引起鼻子、喉咙和肺部的刺激。执行与油烟机内甲醛有关的所有过程。</li>
	<li>在100毫升异丙醇中溶解300毫克, 制备油红色 O 型溶液。</li>
	<li>把 microwell 盘子从孵化器里拿出来。以下步骤可在无菌环境中执行。</li>
	<li>从井中取出所有介质, 用4% 甲醛固定细胞30分钟。</li>
	<li>在孵化时间, 准备油红色 O 工作解决方案。工作解决方案包括6件油红色 O 型股, 4 份蒸馏水 (dH2o)。混合好, 让坐在室温下20分钟, 过滤前使用0.2 µm 过滤器单元。工作解决方案只稳定2小时。</li>
	<li>吹打从水井中取出所有甲醛。</li>
	<li>用60% 异丙醇洗涤细胞, 并在继续前让水井完全干燥。</li>
	<li>添加50µL 的油红 O 工作溶液, 每井和孵化在室温下10分钟。</li>
	<li>除去油红色 O 溶液, 并立即添加 dh2o 用 dh24 次清洗, 然后再在光显微镜下查看。</li>
</ol>4. 鉴别基因特异抗体的染色
<ol><li>通过溶解80克氯化钠、2克氯化钾和30克三基到 dH2O (pH 8) 的1升, 制备三缓冲盐水 (tb) 的库存溶液。使用 dH2o 在室温下存储, 以稀释1:10 来准备工作溶液。</li>
	<li>准备 immunobuffer (IB) (1% 胎牛血清 (血清) 在 TBS)。标签15毫升离心管与日期和存储在4摄氏度。IB 可用于1星期从日期。</li>
	<li>用200毫升磷酸盐缓冲盐水 (PBS) 溶解0.5 克酪蛋白和0.195 克叠氮化钠, 制备0.25% 酪蛋白阻滞剂。通过搅拌过夜, 完全溶解成分。整除15毫升离心管, 并贮存在-20 摄氏度的冷冻机中。解冻酪蛋白溶液可保持在4°c 1 月。</li>
	<li>通过稀释 DAPI 1:200 的 DAPI 溶液, 将其储存在4摄氏度, 免受光照。</li>
	<li>通过在无菌 PBS 中溶解硫柳汞, 制备库存硫柳汞溶液 (10毫克毫升-1)。存放在4°c 和稀释适当的数额到0.4 毫克 mL-1使用。</li>
	<li>固定和 permeabilize 的细胞与冰冷甲醇 (96 井板) 5 分钟洗涤细胞一次与200µL 的 tb。</li>
	<li>块与50µL 0.25% 酪蛋白在室温下10分钟, 并洗涤一次与 tb。</li>
	<li>添加50µL 的主要抗体稀释在 IB (见表 1的抗体细节) 和孵化与眼睑关闭为1小时。</li>
	<li>洗一次与200µL 的 tbs, 然后3倍与 tbs 5 分钟的温柔摇摆。</li>
	<li>添加50µL 的二级抗体稀释在 IB (参见表 2的抗体细节) 与 1:2000 DAPI 在每个, 孵化与盖子闭合 1 h. 用箔盖板以防止光漂白。</li>
	<li>用 tbs 洗一次, 然后两次与 tb 15 分钟的温柔摇摆。</li>
	<li>卸下所有的 tb 并添加200µL 的硫柳汞溶液。</li>
	<li>储存板在4摄氏度, 保护免受光, 直到成像。</li>
</ol>5. 使用高通量显微镜的成像细胞
<ol><li>利用随附软件控制的高含量筛查显微镜, 分析染有基因特异抗体和荧光共轭二次抗体 (免疫荧光) 的细胞。</li>
	<li>将 microwell 板加载到显微镜阶段。在车牌采集控制中, 选择中等幅度放大图像 (10X 物镜)。确保图像是从每个井的中心拍摄的, 每个站点之间有50µm 间距, 以确保一个单元格不会出现在两个相邻字段中。</li>
	<li>选择要图像的水井。</li>
	<li>选择适当的通道组合、曝光时间和 z 偏移参数。有关每个筛选器 (辅助表 2) 的详细信息, 请参阅表 3 。</li>
	<li>使用采集向导获取检测 (图像自动存储到数据库服务器上)。</li>
	<li>使用备用通道组合获取沾有油红色 O 的印版 (补充表 3)。</li>
</ol>6. 分析获取的图像
注意: 对数据库服务器的远程访问能够快速、轻松地评估图像分析软件所获得和使用的图像。
<ol><li>在分析软件上打开单元格评分模块。</li>
	<li>使用 DAPI 通道 (核标记) 和感兴趣的段核来检测细胞核, 其大小和信号强度高于背景。</li>
	<li>使用 FITC 通道检测 FABP4 信号。区分细胞与用户定义参数的大小和信号强度以上背景, 以选择所有 FABP4 的积极区域在每个图像。</li>
	<li>在分析软件上打开 Transfluor 模块, 分析沾有油红色 O 的板材。</li>
	<li>指定油红色 O 染色区域的大小和强度为 "坑"。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.+Part+II.">Image processing with ImageJ. Part II.</a> Biophotonics Int. 11 (7), 36-43 (2005).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7 (10), R100 (2006).</li></ol>

作者声明没有利益冲突。

本文阐述了 FABP4 immunolabelling 对间充质基质细胞的成熟脂肪干细胞进行准确检测和定量量化的效用和优越性。FABP4 能够检测到一个沿袭特定蛋白3,4,5在成熟脂肪细胞中表达的变化, 与其他常用染料的标签大分子变化13,14如油红色 O15、尼罗河红色16和苏丹黑色17。FABP4 immunolabelling 允许可视化和分析细胞形态学的变化。这是一个独特的优势比以前描述的方法使用定量反向转录 PCR (RT-qPCR), 分析变化的成绩单水平没有任何可视化5,18,19 、20或流式细胞术, 要求单元格处于悬浮20中, 或需要细胞裂解以隔离蛋白质1920的西方印迹。FABP4 immunolabelling 是稳定的固定细胞, 不泄漏出在溶液中, 是一个胞浆蛋白当标签在一个黏附分子层的细胞, 将重叠与核允许容易的可视化和增加准确性的自动化分析。
高含量的筛选是有利的, 因为它是快速, 准确, 重现性和客观。它提供了一个成本有效使用试剂和研究员时间的平台, 因为图像采集和分析需要最少的手工操作, 并且依赖于仪器的自动处理。有几个因素被认为对高含量筛选化验的准确性和重现性至关重要31。抗原表达的量化依赖于图像质量。为了获得成功的图像分析, 每个频道的图像必须集中在一个灰度值的最佳动态范围内 (自动曝光设置是理想的)。不集中或饱和的图像导致错误的结果。
本文所述方法的一些潜在限制包括以下内容。对专门的成像设备和分析软件的要求。在本文的范围之外, 可使用替代的开源软件选项 (例如, ImageJ32、33、斐济34、CellProfiler32、35) 来执行类似的图像分割。任务;但是, 他们需要的技能, 以下载和定制的宏可用在线或编写宏/命令, 他们的特定分析管道。所有自动成像分析管道的固有特性是一个背景噪声级别。这主要归因于碎片、图像质量差或分析错误, 强调需要仔细取样准备和仔细设置成像和分析平台。发现小鼠的 FABP4 标签检测未分化的祖细胞30;虽然本研究中的控制 (包括未分化细胞) 缺乏特定的 FABP4 染色。这不足以检查在每一个生物背景下, 在使用该检测的每个生物学环境中检验未分化祖 FABP4 表达的必要性。
为了在 FABP4 标签和油红色 O 染色之间进行有效的比较, 从图像分析中得出的输出测量需要与生物学相关。因此, 测量, "% 阳性细胞" 被用于 FABP4, 和 ' 染色面积每细胞 ' 被用于油红色 O。值得注意的是, 该措施 '% 阳性细胞 ' 没有用于油红色 O 标记细胞在我们的研究, 因为它是不可能准确地将标签的脂肪滴归因于一个给定的核;脂肪滴在细胞体的大小、数量和分布上相差很大, 很少与细胞核重叠。不同组织来源的细胞之间存在油红色 O 染色变异;虽然 the% 阳性细胞测量不适合目前的研究, 另一组已成功地使用它来量化从人类腺体干细胞衍生的脂肪细胞22 , 其中油红色 O 染色出现的一个大脂肪滴, 横跨细胞质和细胞核重叠, 使数据 as% 阳性细胞呈现。
相比之下, FABP4 immunolabelling 的形态学是一致的脂肪细胞来自不同的组织来源的范围23,24,25。在能够分化的文化中, 量化细胞比例的能力有许多应用。成人间充质基质细胞对多种治疗用途具有重要的前景。临床翻译中出现的一个重要问题是, 在26之间、隔离站点27、28和隔离方法之间, 这些细胞之间的能力固有变化12和扩展单元格编号12用于治疗使用。以前已经表明, 黏附的基质细胞的文化经常是异质的, 有些细胞可以分化, 一些不是12,17 , 因为我们的 FABP4 化验显示了 ASC 文化。像这样的化验, 结合浓缩技术, 将有助于确定异种文化中的亚群体, 能够对谱系特异分化。有一个标准化的, 可量化的化验, 如这种 FABP4 化验提供了一个简单的方法来比较所有这些变量, 并有可能评估治疗结果内和之间的临床试验。
FABP4 的量化在半自动化的方式, 使客观和重现性的分析, 在许多捐助者的案例和样本。此外, 由于标签是细胞质, 它可能被用来分析肥大 (脂肪细胞大小的扩大) 除了增生 (脂肪细胞数量增加), 这是相当重要的区别, 在肥胖研究, 其中肥厚与肥胖个体的脂肪功能障碍密切相关21。肥胖症的流行已经激发了对能够控制脂质贮存的药物的大量兴趣。这种 FABP4 的检测还提供了一个简单的读数, 以筛选数以千计的化合物, 以抑制脂质积累的能力。

国际细胞治疗学会 (ISCT) 确定多能间充质基质细胞的关键要求之一是细胞必须具有分化成脂肪、成骨和软骨血统的能力。1. 传统的方法测量分化成这三血统依赖于检测大分子产品使用化学染料1。染料, 如油红色 O (在细胞中染色脂肪滴在已经历成脂), 是廉价和易于使用;然而, 当间充质细胞分化为各自的谱系2时, 它们无法检测出基因表达的具体变化。在这里, 我们已经开发了一种分化检测, 量化的蛋白质表达脂肪谱系特定标记, 脂肪酸结合 protein-4 (FABP4)3,4,5。FABP4 最初在小鼠 3T3-L1 脂肪细胞3中发现, 后来被发现在人皮下脂肪组织6中表达。它是一种胞浆蛋白, 它作为一个伴侣来指导细胞的脂肪酸摄取, 并参与了脂肪4的过程。
用于分化检测的前体细胞为脂肪源性间质基质细胞 (陶瓷)7,8。陶瓷与骨髓间充质干细胞 (BM-MSCs) 有许多性质, 这是成人的主要骨髓间充质干细胞,8,9。在临床应用中, 陶瓷提供了一些优于 BM MSCs 的优势, 因为更大的细胞产量可以从更容易接近的组织来源中分离出来8,9。一个孤立的细胞群需要满足一定的标准来定义为陶瓷。首先, 它们必须在标准培养条件下表现出对塑料组织培养容器的粘附1。它们还必须显示特定的表面抗原表达式1。落后陶瓷的特征是 CD34 的阳性表面抗原表达, CD73, CD90, CD105 的表达低和 CD45 和 HLA-DR10的阴性表达。陶瓷在塑料上培养28天 (黏附纯化陶瓷) 显示 CD73、CD90 和 CD105 的阳性表达, CD34、CD45 和 HLA-DR10的阴性表达。最后, 单元格必须保留区分为各种沿袭的能力1,7,8。
脂肪分化协议诱导上调的 FABP4 表达在其他脂肪细胞谱系基因, 所以我们使用免疫化学可视化 FABP4 蛋白在单元格中, 然后量化 FABP4 表达在单细胞水平使用全自动荧光高含量筛查显微镜。这种方法优于传统染料, 因为它能够高度明确地确认脂肪细胞谱系分化。这种基因特异谱系检测结合高含量筛选方法也可以量化的细胞比例在异构细胞的准备, 能够分化下来的特定血统。在我们的研究中, 我们使用 FABP4 检测来确认细胞培养后新鲜分离陶瓷脂肪分化潜能的丧失。

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Tris Base</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15504-020</td>
			<td style="width:237px;">Tris buffered saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>Merck</td>
			<td>1064041000</td>
			<td>Tris buffered saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Merck</td>
			<td>1049360500</td>
			<td>Tris buffered saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Azide</td>
			<td>Scharlau</td>
			<td>SO0091</td>
			<td>Casein blocker solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Casein</td>
			<td>Sigma</td>
			<td>C7078-500G</td>
			<td>Casein blocker solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS tablet</td>
			<td>Sigma</td>
			<td>P4417-100TAB</td>
			<td>Phosphate buffered saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10091-148</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Select Enzyme (1X), no phenol red</td>
			<td>Gibco</td>
			<td>12563-029</td>
			<td>Cell dissociation enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate (flat bottom)</td>
			<td>Falcon</td>
			<td>FAL353072</td>
			<td>Cell culture equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T75 culture flask, with filter cap</td>
			<td>Greiner</td>
			<td>658175</td>
			<td>Cell culture equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Sigma&#160;</td>
			<td>19278-5ML</td>
			<td>Adipogenic differentiation media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>Adipogenic differentiation media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">indomethacin</td>
			<td>sigma</td>
			<td>I7378-5G</td>
			<td>Adipogenic differentiation media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil-Red O</td>
			<td>Sigma</td>
			<td>O-0625</td>
			<td>Classic stain for adipogenesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropanol</td>
			<td>Merck</td>
			<td>1.09634</td>
			<td>Classic stain for adipogenesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% formaldehyde</td>
			<td>Pierce</td>
			<td>28908</td>
			<td>Cell fixation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Merck</td>
			<td>100983</td>
			<td>Cell fixation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti rabbit IgG alexa 488</td>
			<td>Molecular Probes</td>
			<td>A11008</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thimerosal</td>
			<td>Sigma</td>
			<td>T5125-10G</td>
			<td>Immunocytochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-human fatty acid binding protein 4 (FABP4) antibody</td>
			<td>Cayman Chemicals</td>
			<td>10004944</td>
			<td>Marker of adipogenesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Centrifuge tubes (15mL)</td>
			<td style="width:124px;">Greiner</td>
			<td style="width:119px;">GR188271</td>
			<td>General</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Centrifuge tubes (50mL)</td>
			<td style="width:124px;">Greiner</td>
			<td style="width:119px;">GR227261-P</td>
			<td>General</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">ImageXpress Micro XLS</td>
			<td style="width:124px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>high content screening machine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">MetaXpress</td>
			<td style="width:124px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>high content screening software, version 5.4.01</td>
		</tr>
	</tbody>
</table>

visualization and quantification of mesenchymal cell adipogenic differentiation potential with a lineage specific marker

几种染料目前可用于检测骨髓间充质细胞分化为脂肪脂。染料, 如油红色 O, 是廉价的, 易于使用和广泛利用的实验室分析的脂肪潜力的间充质细胞。然而, 它们并不特定于基因转录的变化。我们已经开发了一种基因特异的分化分析, 以研究何时间充质细胞将其命运转变为脂肪血统。免疫标签对脂肪酸结合 protein-4 (FABP4), 一个血统特定的标记脂肪分化, 使可视化和量化的分化细胞。用96井微板块格式量化脂肪分化潜能的能力, 对许多应用有很好的影响。数以百计的临床试验涉及使用成人间充质基质细胞, 目前很难将治疗结果与此类临床试验之间的关系, 特别是在这方面。这种简单的高通量 FABP4 检测为评估患者源性细胞的分化潜能提供了定量的检测方法, 是比较不同分离和扩张方式的有力工具。这一点尤其重要, 因为越来越多的人认识到在间充质细胞产品中对患者进行的细胞异质性。该方法在高通量药物筛选方面也有潜在效用, 特别是在肥胖和糖尿病前期研究中。

本研究采用3种不同方法测定陶瓷的脂肪分化电位。免疫荧光标记 FABP4, 表明该标记局部化的细胞质的分化细胞, 和标签重叠的 DAPI 标签核 (图 1A)。自动图像分析显示, 早期通道细胞分化组中 FABP4 阳性细胞% 的24.6 倍增加, 较晚期通道细胞增加6.9 倍 (图 1B)。油红色 O 染色是局部的脂肪滴分散在整个细胞质的分化细胞, 和标签很少重叠的 DAPI 标签核;然而从目视检查中, 与早期通道细胞相比, 晚期通道细胞中与油红色 O 脂滴有关的细胞核较少 (图 2A)。自动图像分析显示, 早期通道细胞中每个细胞的油红色 O 染色面积增加了11.1 倍, 后期通道细胞中每个细胞的染色面积增加了53.9 倍 (图 2B)。对两个污点进行了成像, 然后使用该软件中的单元评分 (FABP4) 或 Transfluor (油红色 O) 模块进行量化 (补充图 1, 补充表 2-5)。FABP4 表达式的上调被西方印迹分析证实 (图 3,补充图 5)。所有3种分析方法均显示, 早期通道陶瓷的脂肪分化电位高于晚期通道细胞。脂肪差异不影响每个井的总细胞数 (补充图 2, 3)。然而, FABP4-positive 分化细胞的细胞核大小明显小于晚期通道陶瓷的对照细胞 (补充图 3).
我们证明, 随着细胞在文化中的扩张, 或传代, 在脂肪分化的文化中, 细胞的百分比下降, 与以前发布的数据11一致。必须指出的是, 在本研究中无法控制年龄、体重指数或既往病史的因素, 例如,12 , 可能导致不同捐献者样品之间的变异 (补充表 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57153/57153fig1.jpg"> 图 1: FABP4 immunolabelling 表明, 早期通道细胞具有更大的分化潜能, 而非晚期通道细胞.A) 以 DAPI (核染色) 和 FABP4 为标记的早期和晚期通道、控制和分化细胞的代表性图像与合并和分割图像一起显示。分割图像显示有差异的细胞与绿色覆盖和未分化的细胞与红色覆盖。B) FABP4 表达细胞在早期和晚期细胞脂肪分化显著增加, 但早期通道细胞的分化明显高于晚期通道细胞 (曼惠特尼测试 * 表示 p < 0.05 和 *** 表示 p < 0.001)。所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样品, 电子扫描电镜.<a href="/files/ftp_upload/57153/57153fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57153/57153fig2.jpg"> 图 2: 油红色 O 染色表明, 早期通道细胞具有更大的分化潜能比后期细胞.A) DAPI (核染色) 和油红 O (灰、脂滴染色) 的代表性图像与合并和分割图像一起显示。分割图像描述所有细胞核与绿色覆盖和油红色 O 染色的脂滴与红色覆盖物。B) 在脂肪分化的情况下, 发现油红色 O 染色区显著增加。早期通道细胞与晚期通道细胞相比, 每个细胞的油红色 O 染色面积更大。每个细胞的油红色 O 染色面积 (微米2) 在这里用作脂肪分化电位的量度。所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样品, 电子扫描电镜 (曼惠特尼测试 * 表示 p = < 0.05 和 *** 表示 p = < 0.001)。<a href="/files/ftp_upload/57153/57153fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57153/57153fig3.jpg"> 图 3: FABP4 蛋白表达水平的西方印迹分析区分的早期和晚期陶瓷.FABP4 带的光密度的半定量分析作为总蛋白质装载控制的比率, β肌动蛋白 (ACTB), 表明 FABP4 immunolabelling 在早期的段落明显地增加了, 并且到较少程度晚段落区分细胞.所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样品, 电子扫描电镜 (曼惠特尼测试 * 表示 p = < 0.05 和 *** 表示 p = < 0.001)。<a href="/files/ftp_upload/57153/57153fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>靶抗原</td>
			<td>同种 (克隆)</td>
			<td>物种</td>
			<td>稀释</td>
		</tr>
<tr>
<td>FABP4</td>
			<td>多</td>
			<td>兔</td>
			<td>1:100</td>
		</tr>
</tbody></table>表 1: 原发性抗体
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>靶向抗体</td>
			<td>荧光标签</td>
			<td>物种</td>
			<td>稀释</td>
		</tr>
<tr>
<td>兔 IgG</td>
			<td>Alexa 氟488</td>
			<td>山羊</td>
			<td>1:200</td>
		</tr>
</tbody></table>表 2: 二级抗体
辅助图 1: 图像分析虚拟环境概述.这些染色的细胞使用自动的高通量荧光显微镜成像, 以避免任何偏见的来源。使用 ImageXpress 微 XLS 获取的图像被直接保存到 MDCStore 数据管理器数据库中, 并可通过虚拟桌面连接进行查看, 用户可以使用它们来优化和测试其特定的分析参数。使用专用的 "自动运行" 实例分析图像, 使多个不同的用户可以将 "作业" 发送到自动运行队列。用户可以在 "作业" 完成后通过虚拟实例检索其数据。<a href="/files/ftp_upload/57153/Supplementary_Figure_1.jpg" target="_blank">请单击此处下载此图.</a>
补充图 2: 使用 FABP4 染色板, 比较控制井的总细胞数和用于早期和晚期通道陶瓷的区分井.所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样本, 电子扫描电镜。早期和晚期陶瓷的对照和分化细胞无统计学意义差异。与晚期通道细胞相比, 早期通道细胞每井的细胞都多。<a href="/files/ftp_upload/57153/Supplementary_Figure_2.tif" target="_blank">请单击此处下载此图.</a>
补充图 3: 使用油红色 O 色板, 比较控制井和有区别的井的总细胞计数在及早和晚段落陶瓷.所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样本, 电子扫描电镜。早期和晚期陶瓷的对照和分化细胞无统计学意义差异。<a href="/files/ftp_upload/57153/Supplementary_Figure_3.tif" target="_blank">请单击此处下载此图.</a>
补充图 4: 在以后的段落中, FABP4 阳性的分化细胞的细胞核面积明显小于控制井中的细胞.早期对照和分化细胞间细胞核面积无统计学意义差异。所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样本, 电子扫描电镜. (不配对 t 测试. ** 表示 P = < 0.001)。显示的是。<a href="/files/ftp_upload/57153/Supplementary_Figure_4.tif" target="_blank">请单击此处下载此图.</a>
补充图 5: 西方污点凝胶的图像。红色通道描绘的是β肌动蛋白。绿色通道描述 FABP4 immunolabelling.<a href="/files/ftp_upload/57153/Supplementary_Figure_5.tiff" target="_blank">请单击此处下载此图.</a>
补充表 1: 捐助者的临床病理资料<a href="/files/ftp_upload/57153/Supplementary_table_1.xlsx" target="_blank">请单击此处下载此表.</a>
辅助表 2: 图像设置 (FABP4)<a href="/files/ftp_upload/57153/Supplementary_table_2.xlsx" target="_blank">请单击此处下载此表.</a>
辅助表 3: 成像设置 (油红色 O)<a href="/files/ftp_upload/57153/Supplementary_table_3.xlsx" target="_blank">请单击此处下载此表.</a>
补充表 4: 使用的分析参数表 (FABP4)。单元评分模块。<a href="/files/ftp_upload/57153/Supplementary_table_4.xlsx" target="_blank">请单击此处下载此表.</a>
补充表 5: 使用的分析参数表 (油红色 O)。反流体模块。<a href="/files/ftp_upload/57153/Supplementary_table_5.xlsx" target="_blank">请单击此处下载此表.</a>

Watch this Scientific Journal Video about Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker at JoVE.com

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

传统的评价脂肪分化的方法既便宜又容易使用, 但并不特定于基因表达的变化。我们已经开发出一种用谱系特异标记将间充质细胞分化为成熟脂肪的实验。这种检测方法在基础研究和临床医学中有不同的应用。

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

visualization-quantification-mesenchymal-cell-adipogenic

Research

JoVE Journal

Biology

9.7K 次观看.  The University of Auckland. 传统的评价脂肪分化的方法既便宜又容易使用, 但并不特定于基因表达的变化。我们已经开发出一种用谱系特异标记将间充质细胞分化为成熟脂肪的实验。这种检测方法在基础研究和临床医学中有不同的应用。

视频: Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

科研

生物学

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 ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

操纵小鼠泪腺

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

一个具体的感觉细胞的共聚焦相关和3D电子显微镜

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

MCF7细胞单层中细胞基质粘附面积和细胞形状分布的定量

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins.
Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

与抗原标记阻断病变的复制体遭遇的可视化

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In ...