我们承认哈罗德·德沃夏克博士的有益讨论包括建议尽量固定在37℃煤灰。这项工作是由美国国立卫生研究院授予P01 CA92644和由国家癌症研究基金会的合同支持。

1。胶原镀膜玻璃磁盘细胞培养<ol><li>这里的玻璃盘（直径12毫米）在一个玻璃瓶（4盎司）和高压釜灭菌的光盘。 </li><li>把25毫升70％乙醇在50毫升猎鹰管，并将其放置在细胞培养罩。此溶液可以重复使用多次，直至该溶液的液面下降至20ml。 </li><li>用它来处理的玻璃盘前放置一个锋利的钳用极细点在70％乙醇5分钟。 </li><li>小心取出钳出管，盖上，然后轻轻将乙醇处理钳在管顶空气干燥2分钟。不要让镊子尖在引擎盖碰任何东西。 </li><li>用无菌镊子抢单无菌玻璃盘出来的玻璃瓶，并将其放置在24孔细胞培养板的孔中。重复，直到井所需数量已经填充了光盘。 </li><li>把500微升的牛胶原蛋白溶液（50纳克/毫升）到每一个包含一个玻璃盘，并将其放置在37℃，5％CO 2的细胞培养箱中培养至少30分钟很好。有一个较长的潜伏期没有坏处。 </li><li>收获HUVEC（人脐静脉内皮细胞）或PC3（前列腺肿瘤细胞）已经长大150 MM细胞培养板，通过胰蛋白酶消化并利用其完整的培养基阻止胰蛋白酶的活性。收集细胞入15ml Falcon管中。 </li><li>计数细胞数量，并确保生存能力大于90％。 </li><li>通过在200×g离心10分钟，离心在4℃下沉淀细胞除去上清液。 </li><li>用培养基稀释细胞以10 5细胞/ ml。放置在冰上的细胞。 </li><li>吸出胶原蛋白在细胞培养罩和轻轻地放在500μl的细胞悬浮液以每孔中，其中玻璃圆盘预先涂有胶原蛋白。 5×10 4个细胞/孔在24孔板会给60％置信luency的HUVEC和30％汇合为PC3细胞。注：添加更多的细胞悬液更高的细胞密度。 </li><li>培养的细胞在37℃，5％CO 2培养箱中培养1小时，2小时，4小时，6小时，或24小时来跟踪nanopodia活动和细胞传代。通常情况下，细胞会附着在玻璃盘里的前30分钟，然后开始分化和迁移中的第一个小时，并在下列时间进行细胞间的相互作用和细胞分裂。 </li></ol> 2。细胞固定<ol><li>每个孔将需要500微升4％PFA（多聚甲醛）固定细胞。可商生产的或新鲜的4％PFA中都可以使用。计算需要的基础上制备的井的数量煤灰的量，然后将PFA中15毫升Falcon管中。注意：多聚甲醛是一种可疑的致癌物质。 </li><li>把猎鹰管的聚苯乙烯泡沫塑料的立场，已经到位的37℃培养箱中培养。离开管在孵化器30分钟战m，最高的煤灰至37°C。 </li><li>放置一个加热垫在培养罩并打开它。 </li><li>取出24孔细胞培养板和所述预热的PFA出培养箱，并将其放置在所述加热垫的顶部。 </li><li>用一只手来从井吸出培养基，紧接着用另一只手轻轻滑动，加入500μl的PFA入井通过井边缘。为了更方便地吸出，倾斜培养板在45°左右，倚靠在聚苯乙烯泡沫塑料的立场，使其稳定在那个角度。这将有利于愿望和添加PFA解决良好，没有文化干扰细胞。这是为了保持细胞活动的原来的细胞结构中最关键的步骤。 </li><li>重复上述过程，直到所有的井用玻璃盘已经收到煤灰。注意：需要改变的还有预现有的解决方案中的所有步骤将适用同样的愿望手续。 </li><li>将板在37℃下5分钟。 </li&gt; <li>就拿板回文化引擎盖和倾斜对泡沫塑料按照步骤2.5中所述。用一只手从井里轻轻地转移到煤灰收集管;用另一只手来之后立即放置至少500μl的室温PBS中的井。之后所有的孔都被冲走，返回和在每一个重复的PBS洗涤一次好。清空收集的煤灰成为危险废物的容器。 </li><li>通过加入2％胎牛血清的PBS来制备ICC封闭缓冲液。 0.04％的叠氮化钠（20％原液稀释）加入作为防腐剂。注意：叠氮化钠是一种有毒化合物。缓冲器可以存储在4℃的很长一段时间没有细菌污染。 </li><li>随着文化板块仍偏向反对立场，再次循环通过每个孔抽吸去除PBS中，随即加入500微升的ICC封闭液。细胞现在已经准备好进行免疫荧光染色。如果需要的话，固定的细胞可以被存储在4℃的冰箱中一周不显著损失TM4SF1蛋白质。 </li></ol> 3。 Nanopodia的TM4SF1免疫荧光染色<ol><li>在各孔中，替换500μl的ICC封闭缓冲液与含有0.01％的Triton X-100的一种新的封闭缓冲液，并让它在室温下静置1小时。不要使用比0.03％的Triton X-100更高，因为它会从细胞膜中删除TM4SF1。 PBS洗涤细胞从步骤2.10，可直接移动到包含海卫一的封闭液，如果染色准备马上开始。 </li><li>稀释初级抗TM4SF1（或抗CD9）抗体以0.5微克/毫升在ICC封闭缓冲液。 </li><li>在每个孔中，取出ICC/0.01％的Triton缓冲液，并用300微升的反TM4SF1-抗体溶液替换。孵育2小时，在室温或离开过夜，在4°C。 </li><li>每个不小于500微升PBS洗好。重复3次;每次给于乐AST 5分钟的孵育时间。 </li><li>备中的第二抗体和鬼笔环肽溶液ICC封闭缓冲液中，用1000倍稀释的Alexa 488标记的驴抗小鼠二抗（2μg/ ml的终浓度）和1,000×稀释的鬼笔环肽（50纳克/毫升最终浓度）。 </li><li>除去PBS中，并加入300微升的二级抗体和鬼笔环肽溶液的各孔中，孵育2小时或离开它过夜，在4°C。 </li><li>重复PBS洗涤步骤与至少一个每次洗涤5分钟温育。在最后一次洗涤，每孔在PBS中1小时离开。 </li><li>降防褪色安装媒体上的载玻片一滴（约10微升）。 </li><li>弯曲的针头90°19 G 1½通过在坚硬的表面轻轻按压笔尖。用一只手握住弯曲的针，轻轻抬起玻璃盘从井里，并用另一只手用锋利的钳抓住光盘。 </li><li>把玻璃盘＆＃160;面朝下让于载玻片上的细胞相接触的安装介质。轻轻地放在玻璃盘，让它在室温下暗处干通宵。 </li><li>继续执行图像免疫荧光染色nanopodia，或存放在一个幻灯片框的幻灯片在-20°C。该幻灯片将在-20°C保持可见数个月</li></ol>

<ol>
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没有利益冲突的声明。

Nanopodia薄细胞膜通道，牢牢附着基质通过TMED，可以从一个极化移动细胞感知环境并介导细胞间的相互作用2,3延伸超过100微米。 Nanopodia坚持以矩阵如此坚定地认为残留物留下的细胞移开（ 图1和图2）。 Nanopodia从而使我们能够研究细胞如何感知他们的环境，并确定细胞运动的路径，基因表达或药物治疗是如何变化的运动。然而，由于这些薄（100-300微米宽度）膜结?...

在极化运动，动物细胞延长了多种从它们的表面活力，细胞膜突起，其中包括丝状伪足，伪足，回缩纤维，和褶边1。最近加入到这个名单是nanopodia，一个新认识的类型的薄（100-300微米，直径），伸长（可达50〜100微米长）投影膜提供的膜通道的F-肌动蛋白的结构，如丝状伪足的延伸和回缩纤维，而且染色水平与TM4SF1（跨膜-4-L-6家族-1）中培养的内皮细胞和肿瘤细胞2,3。 TM4SF1是用四旋状的拓扑结构，最初被称为发现，即该分子是内皮细胞的生物标记物中起着内皮细胞增殖和迁移2,3必不可少的作用前的肿瘤细胞抗原4的蛋白质。免疫荧光染色发现TM4SF1被定位于perinuc利尔囊泡和到质膜，并富含TM 4SF1ënriched微ðomains（TMED）。 TMED锚nanopodia到矩阵和存在于nanopodia 1-3 TMED /微米长度的规则排列的条带状花纹。 Nanopodia通常是从一个细胞的前领先，并延长电池的极化为移动中尾随后面。由于TMED的牢固附着的性质，nanopodia无法缩回到细胞，因为它移动远离;弃nanopodia残基从而描绘出蜂窝移动路径。 Nanopodia提供的膜通道的F-肌动蛋白的扩展和回缩，且细胞间的相互作用和通信2,3的网站。这些特点意味着nanopodia提供研究细胞分化，对环境的感知细胞，测定细胞运动的路径和方向，和细胞间的相互作用过程中的F-肌动蛋白装​​配背后的机制一个独特的机会，一个ND通信。 由于TMED的高度疏水性质和nanopodia的薄和脆弱的膜性质，需要特别注意采取以保存TMED和nanopodia。 nanopodia和去除TMED由共同的实验室方法的破坏是一个可能的原因，只有63出版物自1986年1它的首次发现和完全缺乏TM4SF1富集在100-300微米微区上的知识出现在TM4SF1细胞表面，直到TM4SF1的内皮细胞在2009年2报告。 常规的免疫染色方法通常使用像乙醇，甲醇，丙酮或有机溶剂来修复细胞，并使用0.1％或更高的Triton X-100浓度通透细胞5。这里所描述的研究实现了三个重大变化，以常规方法揭示TMED和nanopodia：（i）使用37℃的4％PFA和修复细胞的3776，C培养箱，（ⅱ）申请温和室温下PBS中洗涤，并且（iii）使用低于0.03％的Triton X-100只简要地通透细胞除第一抗体之前，如曲通X-100 0.03％更高将提取TM4SF1。 所有四旋蛋白形成细胞膜上的6微区和在内皮细胞和肿瘤细胞中某些2,3共定位与TMED。如像CD9，CD81，和CD151四旋蛋白被广泛表达，在这里所描述的染色方案可以扩展到那些缺乏TM4SF1对nanopodia功能的研究很多不同的细胞类型。

<table border="0" cellpadding="0" cellspacing="0" width="1597">
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			<td height="21" style="height:21px;width:323px;">glass discs&#160;</td>
			<td style="width:160px;">Fisher Scientific</td>
			<td style="width:133px;">12-545-82&#160;</td>
			<td style="width:983px;">12-mm diameter</td>
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			<td height="21" style="height:21px;width:323px;">glass jar</td>
			<td>Fisher Scientific</td>
			<td>02-912-310</td>
			<td>Certified Clean Clear Glass Straight-Sided Jars, 4 oz.</td>
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			<td height="21" style="height:21px;width:323px;">glass slide</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">12-544-1</td>
			<td>Fisherfinest Premium Plain Glass Microscope Slides</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50-ml falcon tube&#160;</td>
			<td style="width:160px;">BD Falcon</td>
			<td style="width:133px;">352070</td>
			<td>50-ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">15-ml falcon tube with Styrofoam rack</td>
			<td style="width:160px;">Corning</td>
			<td style="width:133px;">430790</td>
			<td>Corning 15-ml PP Centrifuge Tubes</td>
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			<td height="21" style="height:21px;width:323px;">Sharp forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">13-812-42</td>
			<td>Dissecting Extra-Fine-Pointed Splinter Forceps</td>
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			<td height="21" style="height:21px;width:323px;">24-well cell culture plate</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353047</td>
			<td>24-well Cell Culture Plate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">150-mm cell culture plate&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353025</td>
			<td>150-mm cell culture plate&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">19G 1 &#189; syringe needle&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">309635</td>
			<td>19G 1 &#189;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;width:323px;">Name of Reagent</td>
			<td style="width:160px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;width:323px;">ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2701</td>
			<td>Decon&#39;s Pure Ethanol 200 Proof</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">collagen solution&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">354249</td>
			<td>Collagen I, High Concentration, rat tail, 100 mg</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">trypsin/EDTA</td>
			<td style="width:160px;">Cellgro</td>
			<td style="width:133px;">25-053-CI</td>
			<td><a name="RANGE!D15">0.25% Trypsin-EDTA 1X</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">75889 5 LT</td>
			<td style="width:983px;">PBS, 10X Solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PFA (paraformaldehyde)</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">19943 1 LT</td>
			<td>4% in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td><a name="RANGE!D19">Fetal Bovine Serum</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EGM2-MV</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">T8787</td>
			<td>Triton X-100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaN3</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">S2002-25G</td>
			<td>solubilized in water. 4% stock solution and working concentration is 0.4%.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human TM4SF1 antibody&#160;</td>
			<td style="width:160px;">Millipore</td>
			<td style="width:133px;">MAB3127</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human CD9 antibody&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">555370</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">A-21202</td>
			<td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Phalloidin</td>
			<td style="width:160px;">Chemicon</td>
			<td style="width:133px;">90324</td>
			<td>Rhodamine-conjugated Phalloidin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">anti-fade mounting media&#160;</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">P-36931</td>
			<td>ProLong Gold Antifade Reagent with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Buffers and solutions</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">70% ethanol&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td>17.5 ml of ethanol (200 proof) 
			7.5 ml of double deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS (make 200 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20 ml of 10X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">180 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20% Triton X-100 (make 20 ml)&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">4 ml</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">16 ml double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4% NaN3 (make 25 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">1 g of NaN3</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">50 ng/ml bovine collagen solution in PBS (make 5 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Make stock soln. of 50 &#956;g/ml (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">830 &#956;l of Collage I (3 mg)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml PBS&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ICC Blocking Buffer (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">49 ml PBS</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2% FBS (add 1 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">0.04% NaN3 (add 100 &#956;l of 4% NaN3)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml of ICC Blocking Buffer&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 &#956;l of 20% Triton X-100</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Cell</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HUVEC&#160;</td>
			<td style="width:160px;">Lonza&#160;</td>
			<td>C2517A</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx">http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PC3</td>
			<td style="width:160px;">ATCC</td>
			<td style="width:133px;">CRL-1435</td>
			<td><a href="http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7">http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Equipment</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">cell culture hood</td>
			<td style="width:160px;">NuAIRE</td>
			<td style="width:133px;">Nu-425-600</td>
			<td>NU-425 (Series 60) BIOLOGICAL SAFETY CABINET&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">37 &#176;C, 5% CO2 cell culture incubator&#160;</td>
			<td style="width:160px;">CellStar</td>
			<td style="width:133px;">QWJ300DABA&#160;</td>
			<td>Cellstar CO2 Water Jacketed Incubator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">heating pad&#160;</td>
			<td>K&#38;H Manufacturing</td>
			<td style="width:133px;">1020</td>
			<td style="width:983px;">K&#38;H Lectro Kennel Heated Pad with Free Fleece Cover (http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">centrifuge</td>
			<td style="width:160px;">Sorvall&#160;</td>
			<td style="width:133px;">T6000B&#160;</td>
			<td>Sorvall T6000 (B) Benchtop Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
	</tbody>
</table>

nanopodia - thin, fragile membrane projections with roles in cell movement and intercellular interactions

贴壁培养细胞保持极化状态，支持移动和细胞间的相互作用。 Nanopodia是薄的，细长的，主要是丝状肌动蛋白阴性的内皮细胞和癌细胞膜突起，可以通过TM4SF1（跨膜-4-L-6家族-1）的免疫荧光染色进行可视化。 TM4SF1集群在100-300微米直径TMED（TM 4SF1Énriched微Ðomains）含有3到多达14个独立的TM4SF1分子。 TMED间歇地沿着nanopodia以1至3％μ米TMED并牢固地固定到nanopodia矩阵具有规则间距。这使得nanopodia从偏振内皮细胞或肿瘤细胞的前前或后后方延伸超过100μm，且使膜的残基上MATR被留下九时电池移开。 TMED和nanopodia被忽视了，因为他们的极端脆弱性和敏感性，以温度。常规洗涤和固定破坏的结构。 Nanopodia被直接固定在多聚甲醛（PFA）在37℃下保存，然后短暂暴露于0.01％的Triton X-100的染色之前。 Nanopodia在细胞生物学中打开新的局面：他们承诺将重塑我们的细胞如何感知他们的环境的认识，检测和识别其他细胞的距离，启动细胞间相互作用的密切联系，以及参与运动，增殖的信号传导机制，以及细胞细胞通讯。被用于研究TM4SF1衍生nanopodia开发的方法可以是用于通过经典四旋蛋白，特别是广泛表达CD9，CD81，和CD151的机构​​形成在其它类型的细胞nanopodia的研究是有用的。

对于第1步： 如果细胞（如内皮细胞，并在本研究中使用PC3）都能够正常生长，细胞会附着在30分钟内的胶原蛋白涂层的光盘后，他们是种子，分化，成为移动不久之后，排在他们的道路的延伸nanopodia运动。 图1A和1B分别显示了极化和脐静脉内皮细胞的增殖。 图2A显示极化PC3细胞在移动状态。 对第2步： <p class="jove_content...

Watch this Scientific Journal Video about Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions at JoVE.com

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Nanopodia很瘦但很脆弱的膜通道，向​​上延伸至100微米的细胞的领先的前面或后面尾随和检测的细胞环境。直接固定在较高的Triton X-100浓度为37℃，轻柔洗涤，并避免如乙醇，甲醇，丙酮或有机溶剂，并须遵守这些细胞结构。

Nanopodia  - 薄，易碎膜预测与细胞运动和细胞间的相互作用角色

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

nanopodia-thin-fragile-membrane-projections-with-roles-cell-movement

Research

JoVE Journal

Biology

10.2K 次观看.  Harvard Medical School.  Nanopodia很瘦但很脆弱的膜通道，向​​上延伸至100微米的细胞的领先的前面或后面尾随和检测的细胞环境。直接固定在较高的Triton X-100浓度为37℃，轻柔洗涤，并避免如乙醇，甲醇，丙酮或有机溶剂，并须遵守这些细胞结构。 

视频: Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

科研

生物学

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

实时成像检测方法的评估钙的作用<sup&gt; 2 +</sup&gt;和鞘磷脂酶在成孔毒素伤口的修复

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

闪存和冻结：一种新的技术来捕获膜动力学与电子显微镜

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

中子自旋回声光谱学作为脂膜动力学和膜-蛋白相互作用的独特探针

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

从成年、幼年、幼虫和胚胎斑马鱼中分离心脏和血管平滑肌细胞以进行电生理研究

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...