作者要感谢的跨学科创新计划项目，伊利诺伊大学，授予12035。 SK是由美国国家科学基金会（NSF）资助的格兰特在UIUC 0965918 IGERT：培养下一代的研究人员在细胞和分子力学和生物纳米技术。

制造和官能化变刚度与嵌入的细胞培养表面附近的荧光微球的PA凝胶。 1，功能化的顶级玻璃盖玻片<ol><li>清洁玻璃盖玻片（＃1.0，​​12毫米直径），用肥皂和水洗涤，然后用乙醇除去多余的灰尘。 </li><li>地方玻璃盖玻片Â磨碎的表面（ 即移液管尖端支架）上，使得它们不接触，以便易于与盖玻片相互作用。 </li><li>外套盖的整个表面上滑动和聚-D-赖氨酸（0.1毫克/毫升）1小时（ 图1A）。 </li><li>在此期间，执行1:10,000稀释0.1微米直径的胶体溶液中，红色荧光微球与去离子（DI）水，得到的每20 平方微米约1微凝胶上表面上的粒子密度。参见图2对不同稀释度的结果。这dilution可以进行修改，以满足特定的实验的需要。 </li><li>放置在超声波水浴中稀释的溶液30分钟。 </li><li> 1小时后，用镊子小心地将每个盖玻片，并吹干空气。返回干盖玻片的碎面。 </li><li>拆下超声波浴和吸管150微升稀释的胶体溶液到每个盖玻片。离开10分钟（ 图1B）。 </li><li>用镊子小心地将每个盖玻片，并吹干空气。返回干盖玻片的碎面店在黑暗中待用。 </li></ol> 2，准备PA的凝胶直接在玻璃底培养皿<ol><li>预热加热板至100℃。 </li><li>铺陈的玻璃底培养皿所需的数字（35毫米盘14 mm微型孔，＃1.0）在一个平面上的通风橱。 </li><li>覆盖每个培养皿中微孔的97％3 - 氨基丙基 - trimethoxys玻璃部藤本植物（3-APTES）进行7分钟进行化学活化。采取谨慎，以避免在3 APTES到塑料中的陪替氏培养皿的表面的无意滴落，以避免在聚苯乙烯的降解。 </li><li> 7分钟后，补培养皿用DI水和处置入废物容器。 </li><li>对每道菜重复步骤2.4的3倍，然后摇晃培养皿中，除去多余的水分。将培养皿在热板上直到玻璃部分是干燥的。 </li><li>从热点板块中取出培养皿中，并返回到一个平坦的表面在通风橱。 </li><li>在化学通风橱中，使0.5％的戊二醛溶液，以及用30分钟将溶液覆盖每个培养皿的玻璃部分。采取谨慎，以避免戊二醛到塑料中的陪替氏培养皿的表面的无意滴落，以避免塑料的降解。 </li><li> 30分钟后，补培养皿用DI水和处置入废物容器，以冲洗和去除glutaraldehyde。 </li><li>对每道菜重复步骤2.4的3倍，然后摇晃培养皿中，除去多余的水分。将培养皿在热板上直到玻璃部分是干燥的。 </li><li>前混合该PA凝胶溶液的组分中，将功能化的载玻片放入化学通风橱中，使得它们容易获得，允许凝胶与玻璃底部的快速夹持混合凝胶溶液后的培养皿中。 </li><li>在一个15毫升的离心管中，混合40％双丙烯酰胺，2％的丙烯酰胺和丙烯酸中以表1中列出的浓度直接连续地（改编自发布协议10），以实现期望的基质的弹性。 </li><li>添加100mM的HEPES，10％过硫酸铵，和TEMED在表1中列出的量对应于所希望的基质的弹性来完成凝胶溶液。 </li><li>立即吸取15微升凝胶溶液涂布在玻璃部分的中心的培养皿。 </li><li>马上拿起官能盖玻片镊子。 <ol><li>翻转的盖玻片上，以便该荧光珠是在与凝胶溶液的一侧进行接触。 </li><li>放置盖玻片上轻轻现已液体PA凝胶，使得官能侧与所述凝胶（ 图1C）接触的顶部。注意：为了获得最佳结果，第二个人建议用于将盖玻片，以避免部分聚合的可能性，同时吸取液体PA凝胶溶液到多个培养皿的作用。 </li></ol></li><li>翻转所有的培养皿上，以协助避免对荧光纳米粒子聚合成凝胶的PA水平较低的重力影响。 </li><li>等待至少35分钟，或直至PA凝胶原液已经明显地聚合在其离心管中。 </li><li>翻转培养皿回来了，并填写他们用PBS以协助移除盖玻片。 </li><li>小心地使用培养皿的玻璃部与覆盖片的轮廓相接触，使用镊子刮盖玻片的外周。直到盖玻片被抛下进行几个循环。取下盖玻片和处置在适当的尖锐废弃物容器。 </li><li>除去所有盖玻片后，荧光珠将已转印到凝胶（ 图1D）。覆盖PA凝胶完全用PBS，放置在培养皿盖子上的每个皿中，在4℃下存储。 </li></ol> 3，功能化的PA凝胶纤维连接蛋白<ol><li>如在已建立的协议11中所述制备下列预混合溶液：浸泡溶液（137 mM氯化钠，5％（V / V）甘油）和2x偶联缓冲液（0.2M 2 - （N吗啉代）乙磺酸（MES），10 ％（V / V）甘油，pH4.5）中。 </li><li>使用真空泵在生物罩从含有PA的凝胶的玻璃底培养皿移除所有PBS中。 </li><li>吸管德浸泡溶液到每个凝胶，使得凝胶被完全淹没。在室温下孵育至少1小时。 </li><li>暖1 -乙基- 3 [3 -二甲基氨基丙基]碳二亚胺盐酸盐（EDC） 和 N -hydroxysulfosuccinimide（NHS）至室温。 </li><li>混合10倍的溶液的EDC（150​​毫米，19毫克/毫升的去离子水）和NHS（250毫米，29毫克/毫升的去离子水）。 </li><li>混合1份10倍的EDC，1份10倍的NHS，3份去离子水和5份2×偶联缓冲液中。 </li><li>使用真空泵在生物机罩以除去浸泡溶液。确保所有的液体从凝胶表面除去。 </li><li>添加足够的NHS / EDC溶液，以覆盖凝胶表面并填充培养皿（150-250微升）的玻璃底井。孵育在室温下在黑暗中30分钟。 </li><li>解冻纤连蛋白在室温下。一旦解冻，混合灭菌去离子水来创建一个50微克/毫升纤维连接蛋白的解决方案。 </li><li>使用真空泵在生物罩移除NHS / EDC SOLU化。确保所有的液体从凝胶表面除去。 </li><li>加入150μl的纤维连接蛋白的解决方案，以每个胶。在室温下孵育35分钟，以允许为附着纤连蛋白。 </li><li> 35分钟后，PBS添加到各培养皿中，并储存在4℃下进行最多2周。 </li></ol> 4，牵引力试验<ol><li>温暖的细胞培养基，PBS和胰蛋白酶37℃水浴中。 </li><li>冲洗凝胶5倍用无菌PBS，吸出PBS漂洗中之间，并留下包括在引擎盖。 </li><li>加入1 ml胰蛋白酶每为 25cm 2至含有烧瓶中的细胞。之后将细胞从烧瓶中抬起，稀释与细胞介质的胰蛋白酶和计数细胞。基于凝胶的表面积，确定了最终的细胞接种3000细胞/ cm 2的密度为每凝胶所需的细胞数。稀释或浓缩悬浮液，使150微升的细胞介质混合物中含有细胞的这个数字。分装150微升细胞suspens的离子对每个凝胶。 </li><li>将装有细胞在培养箱中30分钟，在陪替氏培养皿。然后，小心地取出培养皿，并填补了培养皿与媒体（约2毫升）的其余部分，使菜的表面完全被淹没。 </li><li>放置在培养皿放回孵化器，直到图像采集。 </li><li>准备显微镜和数据采集系统成像：将40倍的水浸泡的目的，将微分干涉对比（DIC）棱镜，选择mCherry或同等荧光灯滤光片，并打开室内环境。 </li><li>当成像准备，从孵化器中删除一个培养皿中，并轻轻将它放在显微镜阶段。取出培养皿盖子DIC成像。 </li><li>找到一个单细胞，并捕获DIC小区的单幅静止图像。 </li><li>无需移动显微镜载物台，切换拍摄模式，以荧光。专注于荧光微球和REC奥德微球的图像。 </li><li>小心地从培养皿用移液管除去细胞培养基，并添加0.05％胰蛋白酶-EDTA。 </li><li>图像中的细胞后的细胞在微球具有离。 </li><li>利用粒子图像测速（PIV）分析ImageJ的12位移场计算，由于细胞的力量。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

当使用这种技术时，至关重要的是，在胎圈溶液适当稀释，并且将珠粒根据所要求的直径选择，PA凝胶基体的刚度，和正在探讨在所需实验现象的大小规模。 应该注意，当稀释前官能顶部玻璃盖片的珠液服用。在凝胶表面珠粒之间的间隔可以通过改变胶体溶液的稀释因子来改变。稀释该溶液过多会降低珠耦合到所述盖片的数量，并最终降低了凝胶珠的数量。稀释该溶液太少可...

活细胞与当地环境中的机械作用已普遍被使用的PA凝胶的研究。这些底物依赖于德姆博和王1997年1，其中之一衬底的主要优点建立一个简单的，良好表征的协议是，它们的刚度可通过改变凝胶溶液的特定成分的浓度进行调整。这提供了一个理想的平台来研究细胞的不同刚度的环境互动。当PA凝胶涂覆有细胞外基质（ECM）蛋白，细胞粘附到它们，从而产生动力。作为细胞力的结果，所述凝胶变形作为弹性体。该变形取决于由细胞和凝胶的弹性性能所施加的力的大小。各种研究都采用PA凝胶研究细胞牵引力。 在PA的凝胶制造一种变体中，荧光微球（珠）被嵌入吨hroughout凝胶定量细胞牵引力上刚性不同2的凝胶。当细胞施力，珠取代从以下凝胶变形的初始位置。变形场是从个体压边位移的测量。这个变形场是利用具有弹性的理论和凝胶来计算牵引力的弹性性能。这些测量提供洞察力，如何细胞的机械感，并与他们的局部微环境3交互。 在许多广泛使用的PA硅胶制作协议，将珠混杂在整个液态，未聚合状态的PA凝胶。一个完全聚合的PA凝胶含有在其整个体积荧光珠。当计算单元的牵引力，珠粒最凝胶表面（细胞 - 基底界面）附近进行监视。这些珠的位移被假设为发生在细胞培养表面为简单起见，在该力计算中ñ。的凝胶的深度范围内的珠粒的实际位置被忽略。然而，在弹性介质（如PA凝胶），珠接近的点的力的应用将移动超过胎圈即进一步远离点。因此，治疗的一个点的位移（在胎圈的位置）从表面作为在蜂窝牵引力的低估的表面结果远侧。错误的程度取决于从表面的胎圈之间的距离。不能在没有加强筋的位置的知识，估计误差。 需要有一个简单的方法来限制​​珠非常接近的细胞培养表面已经解决了一些技巧。一种方法是增加的小珠的密度在整个凝胶，使得有足够数量的小珠在顶端焦平面测量表面非常接近运动。另一项技术涉及建筑共聚焦成像室活细胞成像，使得从光仅在最上面的焦平面珠被收集4。不同的方法，包括覆盖一层极薄包含在已经聚合凝胶珠顶无珠5个 PA凝胶。每一个这些技术中的一个缺点是，在小珠的凝胶内的精确位置是未知的。这引入了误差成珠的位移场，因此细胞力的计算的计算。另一种技术涉及的珠偶联到用磺基SANPAH 6的已聚合的PA凝胶的顶部表面。这种技术保证了珠确实只对PA的凝胶的顶部，但是它们所嵌入在凝胶中的深度的程度是未知的。这可能会创造一个局部地形的细胞，这可能会改变细胞的行为，因为以前的工作已经表明，细胞可以感知力几微米的距离7。最近，图案PA凝胶，1微米直径为f的技巧在正规化阵列luorescent纤连蛋白点标记成立8。在这种情况下，荧光标记物的深度是已知的，并且基本上是零，如纤连蛋白图案被间接地印刷在凝胶表面上。然而，这种方法不能提供连续的环境在其上的细胞可以附着，作为ECM的蛋白被限制为1微米直径的圆点。一种充分整合追踪珠PA凝胶内，并限制他们到一个已知位置的表面非常接近的方法还有待建立。 在这里，我们开发了一种技术，亚微米到微米直径的荧光珠限制为焦平面非常接近的PA凝胶内的细胞培养表面。凝胶通常是固化夹在2片玻璃板之间的未聚合的液体凝胶溶液。一板的被官能化，使得该凝胶牢固地粘附到它。另一种是未官能化的凝胶固化后除去。我们修改这个可移动的玻璃苏通过用一层珠涂布它rface。在夹着官能和胎圈包被的玻璃表面之间的液体凝胶，珠转移到凝胶，同时它被固化。这限制了小珠“一体化的距离进入内1.6微米的表面的凝胶。玻璃底培养皿被用作在其上的凝胶进行固化的粘合表面。以形成聚合期间平顶凝胶表面，圆形盖玻片用于夹心与玻璃底培养皿中的凝胶。之前的凝胶制造，顶部玻璃盖片上涂有聚-D-赖氨酸（PDL），产生一个正的表面电荷。 PDL中与压缩空气吹走，并且珠在水中的溶液被沉积在盖玻片上。我们利用羧化荧光微球，其带有负电荷，并与通过用PDL创建的带正电荷的表面相互作用。用压缩空气，单层吹珠溶液脱盖玻片后珠保持静电耦合到干盖玻片。 PDL中涂层不影响玻璃的凝胶表面的胶粘性，因为载玻片未损坏并从PA凝胶充分完整移除。 在玻璃底培养皿是由粘附通过治疗97％3-氨丙基trimethoxysliane和0.5％戊二醛。所需的刚性PA的凝胶是由通过标准程序9混合适当的双丙烯酰胺和丙烯酰胺的含量产生。凝胶溶液的液滴被吸至玻璃底培养皿中。含珠的玻璃盖片是用来夹持与培养皿的凝胶。当凝胶固化时，顶盖滑动除去留下嵌入在PA的凝胶内为1.6μm的表面上的小珠。

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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Reagents</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">97% 3-Aminopropyl-trimethoxysliane (APTES)</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">70% Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>111-30-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1M HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td>83264</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">40% Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">2% Bisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">0.1 um Fluorescent microspheres (mCherry)</td>
			<td>Invitrogen</td>
			<td>F8801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fibronectin, Human, 1mg</td>
			<td>BD Biosciences</td>
			<td>354008</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ammonium Persulfate</td>
			<td>Bio-RAD</td>
			<td>161-0700</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Tetramethylethylenediamine (TEMED)</td>
			<td>Bio-RAD</td>
			<td>161-0801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Poly-D-Lysine</td>
			<td>Millipore</td>
			<td>A-003-E</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)&#160;</td>
			<td>Thermo Scientific</td>
			<td>22980</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">N-hydroxysulfosuccinimide (NHS)</td>
			<td>Thermo Scientific</td>
			<td>24500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">.05% Trypsin-EDTA (1X)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NaCL</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Acrylic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>147230</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7757</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Materials</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">35 mm Glass bottom dish with 14 mm micro-well #1 cover glass</td>
			<td>In Vitro Scientific</td>
			<td>D35-14-1-N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glass cover slips, 12 mm diam.</td>
			<td>Ted Pella, Inc.</td>
			<td>26023</td>
		</tr>
	</tbody>
</table>

a novel method for localizing reporter fluorescent beads near the cell culture surface for traction force microscopy

PA的凝胶已经长期被用作研究，由于容易制造和能力调整自己的弹性性质的细胞牵引力的平台。当基片涂覆有细胞外基质蛋白，细胞粘附在凝胶并施加力，使所述凝胶变形。变形取决于细胞的牵引力和凝胶的弹性性能。如果该表面的变形场是已知的，表面的牵引力可使用弹性理论计算。凝胶变形被嵌入荧光标记物珠粒均匀地进入凝胶常用的测量。探针置换的凝胶变形。凝胶的表面附近的探针被跟踪。报告的这些探头的位移被认为是表面的位移。从表面的深度将被忽略。这种假设在介绍牵引力评估错误。用于细胞力精确的测量，重要的是对于被称为珠的位置。我们已经开发一种技术，其利用简单的化学反应来限制荧光标记珠，0.1和1微米的直径，在PA的凝胶，在1.6微米的表面。我们涂上聚-D-赖氨酸（PDL）和荧光珠盖玻片。 PA凝胶溶液，然后在盖玻片和粘附表面之间。荧光珠在固化过程中转移到凝胶溶液。聚合后，在PA凝胶包含在一个平面上接近凝胶表面荧光珠。

共焦成像被用来确定该珠确实凝胶表面的下方，并在凝胶深度内量化它们的精确位置。一个不同的波长比所述凝胶内的荧光珠沉降的表面上，并嵌入荧光纳米粒子与那些在表面上的距离，使用质心确定算法来计算。在凝胶表面上的加强筋的位置用作用于凝胶，其中细胞在粘附施加力的表面的顶表面上的参考。在两种情况考虑（与红色小珠的表面上，反之亦然凝胶绿色珠）的示意图如图3</stron...

Watch this Scientific Journal Video about A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy at JoVE.com

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

制造含有荧光探针聚丙烯酰胺（PA）的凝胶传统技术涉及的夹附着表面和玻璃载片之间的硅胶。这里，我们表明，涂敷该滑动用聚-D-赖氨酸（PDL）和荧光探针的局部化的探针内1.6微米的凝胶表面。

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

a-novel-method-for-localizing-reporter-fluorescent-beads-near-cell

Research

JoVE Journal

Bioengineering

13.5K 次观看.  University of Illinois at Urbana-Champaign. 制造含有荧光探针聚丙烯酰胺（PA）的凝胶传统技术涉及的夹附着表面和玻璃载片之间的硅胶。这里，我们表明，涂敷该滑动用聚-D-赖氨酸（PDL）和荧光探针的局部化的探针内1.6微米的凝胶表面。 

视频: A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

科研

生物工程

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

无参考牵引力显微镜平台的制造和实施

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

牵引力显微镜研究B淋巴细胞激活

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...