这项工作得到了中国国家自然科学基金 (U1703118) 的支持, 该项目由江苏省高等教育机构 (PAPD) 的优先学术项目开发资助, 江苏 Shuangchuang 项目, 国家重点开放式基金。化学/生物传感和化学计量学实验室 (2016015) 和大分子 (2017kf05) 国家实验室和江苏省特别指定的教授项目, 中国。

注: 一般情况下, 再生过程包括胰蛋白酶消化和漂洗步骤与乙醇和水。消化时间根据所使用的细胞数而变化, 所用细胞的类型和数量可能因实验目的而异。建议使用光学和电化学方法检查再生微晶片, 以优化再生条件。在实验中, 可溶性和不溶性的化学物质可能涉及, 这两种典型的再生程序的例子是详细的。
1. 再生溶液的制备
注: 在无菌条件下准备并处理所有溶液。
<ol><li>准备新鲜的 0.25% (g/v) 胰蛋白酶溶液。胰蛋白酶可以购买或新的准备如下。<ol>
<li>将0.25 克的胰蛋白酶溶解在100毫升的 pH 值7.4 的磷酸盐缓冲生理盐水 (PBS) 中。</li>
		<li>搅拌溶液直到胰蛋白酶完全溶解在 4 oc. 用低速搅拌溶液, 以避免气泡形成。</li>
		<li>在生物安全罩中用0.22 µm 过滤器过滤解决方案。</li>
	</ol>
</li>
	<li>用绝对乙醇制备75% 乙醇。将75毫升绝对乙醇倒入容积瓶中, 加入去离子水, 直到体积达到100毫升。必要时进行进一步的灭菌。</li>
</ol>2. 电子板中 A549 细胞的孵化和增殖
注: 电子板类型16和 L8 都是由排列的金电极 lithographically 打印在孵化井底部。电子板16有16个井, 而电子板 L8 有8个井。电子板16的井是圆的, 而电子板 L8 的井是圆的长方形。每台电子版8井的容积约为830µL, 每台电子板16井约270µL。
<ol><li>A549 细胞培养<ol>
<li>在罗斯威尔公园纪念 Institute-1640 培养基 (RPMI-1640) 中培养 A549 细胞, 在细胞培养皿中补充10% 胎牛血清 (血清) 和1% 青霉素-链霉素。</li>
		<li>通过细胞密度达到75% 然后用 1 x PBS 和胰蛋白酶处理用0.25% 胰蛋白酶溶液在 37 o c 上清洗粘附细胞层, 以1分钟的时间, 将 179 x g 的细胞分离出来以去除胰蛋白酶. 并用重悬10毫升培养基中的细胞进行进一步的培养或其他实验。 注: 在培养基中悬浮细胞的溶液被指定为细胞悬浮缓冲器。</li>
		<li>取出10µL, 用 hemocytometer 计数细胞数。使用 RPMI-1640 培养基进一步稀释细胞悬浮缓冲液, 为实验准备3000万细胞/毫升细胞悬浮缓冲液。</li>
	</ol>
</li>
	<li>
增殖/细胞毒性实验与数据采集
	<ol>
<li>增加50µL (150 µL 为电子板材 L8) 细胞媒介对每台孵化井16并且在生物安全罩保持30分钟为平衡。在 37 oC 中, 在 CO2孵化器中手动插入电子板16在实时单元分析仪中。</li>
		<li>在计算机上启动实时单元分析仪操作程序。在默认的实验模式设置页面上, 选择 "实验模型" 并命名运行检测。</li>
		<li>在其布局页面上, 选择实验中包含的相应井, 并输入编辑框中的单元格类型、编号和药物名称等信息。在其 "计划" 页上, 添加实验步骤, 然后选择每个步骤的运行时间 (例如, 12、24或 48 h) 以及实时数据采集的时间间隔。 注: 在这里, 步骤1和2的间隔是1分钟和15分钟之后。</li>
		<li>保存文件并单击 "开始" 按钮, 测量其单元格索引页上介质的背景阻抗;结果数据将被自动减去。在它的井图页, 所有井曲线可以被参观。在其绘图页上, 加水井和时间细胞指数图将被显示出来。</li>
		<li>对于细胞增殖实验, 首先单击 "暂停" 按钮暂停程序。然后取出电子板, 在室温下添加100µL A549 细胞悬浮缓冲液。把盘子放在生物安全罩里30分钟, 让细胞沉淀到孵化井的底部。</li>
		<li>在实时单元分析仪工作站上手动插入电子版。单击 "开始" 按钮继续进行实验。 注意: 在绘图页上, 分析器在整个实验过程中连续记录单元格索引值或曲线。</li>
		<li>实验完成后, 输入绘图页并打开实验文件。然后选择所有经过测试的孵化井和产生的细胞指数曲线, 在添加药物或改变培养基以减少化验之间的差异之前, 最后一次可以对其进行平均或规范化。在数据分析页上, 可以计算 EC50 或 IC50</li>
	</ol>
</li>
</ol>3. 电子板的再生
注: 对于每个冲洗步骤, 电子版中的溶液完全混合 (5-10 次) 使用吸管。
<ol><li>
案例 1: 对抗癌症药物的细胞毒性评价
	<ol>
<li>在电子版上的种子 A549 细胞, 如第2节所述。</li>
		<li>首先在细胞培养基中溶解抗癌药物阿霉素 (DOX)。慢慢地添加 DOX 到细胞 (DOX 的最终浓度是30µg/毫升) 使用吸管。记录该词, 如第2节所述。 注: 实验完成后, 所用的电子板立即再生。在这种情况下, 由于 DOX 是一种可溶性化学物质, 在协议后的电子板的再生相对容易处理, 不需要额外的步骤。</li>
		<li>
再生
		<ol>
<li>从实时细胞分析仪站取出含有细胞的电子板。将二手电子板放在室温灭菌的生物安全罩中, 用吸管彻底混合培养基。把所有的培养基都吸掉。</li>
			<li>在室温下用200µL 去离子水冲洗电子板3次。</li>
			<li>使用 37 oC, 在孵化器中用0.25% 个新鲜制备的胰蛋白酶 (200 µL/井) 消化电子板上的剩余细胞。当消化完成后, 用吸管将孵化液混合5-10 次, 并在室温下将生物安全罩中的所有溶液吸出。</li>
			<li>分别用200µL 乙醇和200µL 水冲洗电子板, 如下: 去离子水2次, 100% 乙醇2次;75% 乙醇2次;去离子水2次。在无菌纱布或纸巾上反转电子版, 以醒酒在室温下的剩余水分。</li>
			<li>紫外线杀菌的再生电子板在生物安全罩的室温下1-2 小时。 注: 双容量的所有解决方案的再生电子版 L8。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
案例 2: 使用介孔材料进行药物输送。
	<ol>
<li>像以前报告的20一样, 使用以 5.6 nm 的平均孔径作为药物传递矩阵的棒状介孔二氧化硅材料。 注: 在这里, 电子板被用来监测药物释放对细胞的影响。<ol>
<li>将介孔材料添加到电子板各井的孵化液中。由于二氧化硅材料在电子版中不溶于沉淀, 因此应将板材冲洗如下:</li>
		</ol>
</li>
		<li>执行步骤 3.1. 3.1-3.1. 3.3, 如案例1所述。</li>
		<li>在室温下, 在生物安全罩内用200µL 去离子水冲洗微晶片。</li>
		<li>在室温下用200µL 绝对乙醇冲洗微晶片2次。</li>
		<li>在室温下用200µL 75% 乙醇冲洗微晶片。</li>
		<li>在每个井中加250µL 75% 乙醇, 在室温下用密封膜封住电子板。</li>
		<li>旋转 114 x g 2 分钟, 并清空电子版。在无菌纱布或纸巾上反转电子版, 以醒酒在室温下的剩余水分。</li>
		<li>重复步骤 3.2. 5-3. 2.7 次。</li>
		<li>紫外线杀菌的再生电子板在生物安全罩的室温下1-2 小时。 注: 清洗 L8 电子板时, 将所有溶液的体积加倍。</li>
	</ol>
</li>
</ol>4. 再生效果评估
<ol><li>
再生电子板表面的光学特性
	<ol>
<li>使用钢勺仔细拆卸新鲜和再生电子板的孵化井的底部, 其中包含阵列金电极。戴着塑料手套, 小心地用钢勺从电子板上抹去含有微芯片的部分。</li>
		<li>将两个零件放在玻璃显微镜的中心, 并通过拉曼显微镜收集得到的拉曼光谱, 并配有 CCD 探测器21。 注: 光谱仪配备了633纳米激光, 光谱记录在三十年代激光照射的间隔, 波长在500-3500 厘米-1范围内。利用10x 眼透镜和50x 物镜, 用拉曼显微镜获得光学图像。图 1A和图 1C也使用已安装的拉曼显微镜设备获得。</li>
		<li>为了评估在新鲜和再生电子板上抗癌药物摄取后附着细胞的生存能力, 使用共焦激光扫描显微镜对 A549 吸收的 DOX 分子的自发荧光进行监测。<ol>
<li>为了记录细胞吸收 DOX 的自发荧光 (新鲜和再生电子板与 A549 细胞), 使用63X 油目标和485毫微米作为激发波长。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
再生电极的电化学行为 注: 由于商用电子板的手动掩饰是困难的, 不太适合对表面电状态进行检测, 因此使用常规的金/玻碳电极作为试验, 评估了该协议的再生效率。平台上进行电化学阻抗谱 (EIS)。金电极的直径和两个均为3毫米. 电化学分析仪采用三电极系统获得电化学数据, 采用 10 mV 振幅的交流电流信号进行阻抗测量。宽频率范围的100赫到0.01 赫兹。<ol>
<li>测量前, 用抛光布上的氧化铝浆料抛光金电极, 并将其涂成镜面状表面, 然后在水中超声波去除任何颗粒。</li>
		<li>在0.5 米 H2中激活 Au 电极, 因此4。在10毫米氯化钾电解质溶液中获得裸金电极的 EIS 数据, 其中包含 1mM K3[Fe (CN)6], 如所述22。</li>
		<li>根据2.1.2 和2.1.3 准备库存 A549 细胞溶液。在金电极表面上滴下10µL 细胞悬浮液。孵育金电极和经 37 oC 型孵化器中的细胞修饰的 3 h. 获得金电极的 EIS 数据和在10毫米氯化钾电解质溶液中经细胞修饰的, 含 1mM K3[Fe (CN)6]。</li>
		<li>
使用协议重新生成电极并测量 EIS。
		<ol>
<li>将金电极和经0.25% 胰蛋白酶修饰的细胞浸泡在 0.5-2 h 上, 将其浸洗4次。用绝对乙醇轻轻冲洗电极4次。浸入金电极和75% 乙醇中的5分钟, 用75% 乙醇 (4 倍) 轻轻冲洗。</li>
			<li>在含 1mM K3[Fe (CN)6] 的10毫米氯化钾电解质溶液中获得再生金电极和剑桥的 EIS 数据。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

我们总结了几种可用的方法17,23,24,25,26 , 用于在表 1中再生微晶片。基本上, 这些方法涉及相对苛刻的实验条件, 以实现芯片的完全再生, 因为存在着强大的分子-分子相互作用, 如用于 SPR 芯片的免疫复合体。然而, 这些苛刻的实验条件可能对 RTCA 中使用的阵列金微?...

由于其高效和少劳动密集型的实验过程, 在过去十年中, 基于无标签的细胞技术在分析和筛选等方面取得了迅速的增长, 例如蛋白质组学1,2, 药物传递3,等等4,5与传统的生化方法相比, 针对细胞分析, 无标签的实时细胞检测与原型开发的 Giaever 和同事以前6是基于在细胞附着的微晶片表面记录电信号变化的原理, 允许以定量的方式连续测量细胞生长或迁移。根据这一策略, 采用基于电阻阻抗的检测原理的实时细胞电子传感 (RT) 系统介绍了7、8以及最近推出的商用实时单元分析仪 (RTCA)。实验室研究9。
商用实时细胞分析仪主要是从细胞增殖、迁移、生存能力、形态学、黏附力等方面对培养细胞的生理变化进行分析, 以观察其产生的电阻抗的诱发信号。微芯片的表面10,11。这种电信号由分析仪进一步转换成一个无量纲参数, 称为细胞指数 (CI) 来评估细胞的状态。微晶片阻抗的变化主要反映了电极/溶液界面上覆盖细胞的局部离子环境。因此, 细胞分析仪的分析性能很大程度上依赖于核心传感单元, 即一次性微晶片 (即, 所谓的电子板,例如, 96/16/8 井), 这是由阵列金电极lithographically 印在孵化井的底部。金电极装配在一个循环的在线格式 (图 1) 和覆盖的孵化井的大部分表面积, 这允许动态和敏感检测连接的细胞3,12,13 ,14。该词将增加, 在更多的表面覆盖细胞的芯片, 并减少当细胞暴露在毒物导致细胞凋亡。虽然实时细胞分析仪经常用于确定细胞毒性11和神经毒性15 , 并提供比经典端点方法更多的动力学信息, 但一次性电子板是最昂贵的消费品.
到目前为止, 还没有可用的方法来再生电子板, 这可能是由于诸如食人鱼溶液或乙酸等严酷的再生条件涉及16,17,18, 这可能会改变金微晶片的电状态。因此, 在电子板再生过程中, 采用温和有效的方法从金片表面去除粘附细胞和其他物质是可取的。我们最近制定了一项议定书, 目的是利用非腐蚀性试剂再生一次性电子板, 再生芯片的特点是电化学和光学方法19。利用速效和适度的实验试剂, 包括胰蛋白酶和乙醇, 建立了无不良影响的商用电子板再生的一般方法, 成功地应用于再生两种主要类型用于 RTCA 的电子板 (16 和 L8)。

<table border="0" cellpadding="0" cellspacing="0" width="1064">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Rat: Sprague-Dawley</td>
			<td style="width:317px;">Charles River</td>
			<td style="width:263px;">Cat# 400</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">mouse anti-rat CD11b monoclonal (clone OX42)</td>
			<td style="width:317px;">Bio-Rad</td>
			<td style="width:263px;">Cat# MCA275R</td>
			<td style="width:192px;">Panning: 1:1,000; Staining: 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Goat polycolonal anti-Iba1&#160;</td>
			<td style="width:317px;">Abcam</td>
			<td style="width:263px;">Cat# AB5076</td>
			<td style="width:192px;">Staining: 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Rabbit polyclonal anti-Ki67&#160;</td>
			<td style="width:317px;">Abcam&#160;</td>
			<td style="width:263px;">Cat# AB15580</td>
			<td style="width:192px;">Staining: 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Alexa Fluor Donkey anti-mouse 594</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# 11055</td>
			<td style="width:192px;">Staining: 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Alexa Fluor Donkey anti-goat 488</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# A-21203</td>
			<td style="width:192px;">Staining: 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Alexa Fluor Donkey anti-Rabbit 647</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# A-31573</td>
			<td style="width:192px;">Staining: 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Triton-X (detergent in ICC staining)</td>
			<td style="width:317px;">Thermo Fisher</td>
			<td style="width:263px;">Cat# 28313</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparan sulfate</td>
			<td style="width:317px;">Galen Laboratory Supplies</td>
			<td style="width:263px;">Cat# GAG-HS01</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparin</td>
			<td style="width:317px;">Sigma&#160;</td>
			<td style="width:263px;">Cat# M3149</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Peptone from milk solids</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# P6838</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">TGF-&#946;2</td>
			<td style="width:317px;">Peprotech</td>
			<td style="width:263px;">Cat# 100-35B</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Murine IL-34</td>
			<td style="width:317px;">R&#38;D Systems</td>
			<td style="width:263px;">Cat# 5195-ML/CF</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ovine wool cholesterol</td>
			<td style="width:317px;">Avanti Polar Lipids</td>
			<td style="width:263px;">Cat# 700000P</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Collagen IV</td>
			<td style="width:317px;">Corning&#160;</td>
			<td style="width:263px;">Cat# 354233</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Oleic acid</td>
			<td style="width:317px;">Cayman Chemicals&#160;</td>
			<td style="width:263px;">Cat# 90260</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">11(Z)Eicosadienoic (Gondoic) Acid</td>
			<td style="width:317px;">Cayman Chemicals&#160;</td>
			<td style="width:263px;">Cat# 20606</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Calcein AM dye</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# C3100MP</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ethidium homodimer-1</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# E1169</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">DNaseI</td>
			<td style="width:317px;">Worthington</td>
			<td style="width:263px;">Cat# DPRFS</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Percoll PLUS</td>
			<td style="width:317px;">GE Healthcare</td>
			<td style="width:263px;">Cat# 17-5445-02</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Trypsin&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# T9935</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">DMEM/F12</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 21041-02</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Penicillin/ Streptomycin</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 15140-122</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Glutamine</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 25030-081</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">N-acetyl cysteine&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# A9165</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Insulin</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# 16634</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Apo-transferrin&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# T1147</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Sodium selenite</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# S-5261</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">DMEM (high glucose)</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 11960-044</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Dapi Fluoromount-G</td>
			<td style="width:317px;">Southern Biotech</td>
			<td style="width:263px;">Cat# 0100-20&#160;</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Poly-D-Lysine&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# A-003-E</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Primaria Plates&#160;</td>
			<td style="width:317px;">VWR</td>
			<td style="width:263px;">Cat# 62406-456</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Stock reagents&#160;</td>
			<td style="width:317px;">reconstitution&#160;</td>
			<td style="width:263px;">Concentration used</td>
			<td style="width:192px;">Storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Apo-transferrin</td>
			<td style="width:317px;">10 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">N-acetyl cysteine</td>
			<td style="width:317px;">5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Sodium selenite</td>
			<td style="width:317px;">2.5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:25,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Collagen IV</td>
			<td style="width:317px;">200 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">TGF-b2</td>
			<td style="width:317px;">20 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:10,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">IL-34</td>
			<td style="width:317px;">100 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ovine wool cholesterol</td>
			<td style="width:317px;">1.5 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Heparan sulfate</td>
			<td style="width:317px;">1 mg/mL in H2O</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Oleic acid/Gondoic acid</td>
			<td style="width:317px;">Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparin</td>
			<td style="width:317px;">50 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Solutions&#160;</td>
			<td style="width:317px;">Recipe&#160;</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Comments</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Perfusion Buffer</td>
			<td style="width:317px;">50 &#956;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +)</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Douncing Buffer</td>
			<td style="width:317px;">200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Panning Buffer</td>
			<td style="width:317px;">2 mg/mL of peptone from milk solids in DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Microglia Growth Medium (MGM)</td>
			<td style="width:317px;">DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen IV Coating</td>
			<td style="width:317px;">MGM containing 2 &#956;g/mL collagen IV</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Myelin Seperation Buffer</td>
			<td style="width:317px;">9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Mix well after the addition of&#160; CaCl2 and MgCl2</td>
		</tr>
		<tr height="235">
			<td height="235" style="height:235px;width:293px;">TGF-b2/IL-34/Cholesterol containing growth media (TIC)&#160;</td>
			<td style="width:317px;">MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.&#160;</td>
		</tr>
	</tbody>
</table>

regeneration of arrayed gold microelectrodes equipped for a real-time cell analyzer

基于无标签细胞的检测有利于生物化学研究, 因为它不需要实验动物的使用。由于它能够提供更多的动态信息的细胞在生理条件比经典生化检测, 这种无标签的实时细胞分析的基础上的电阻阻抗原理是吸引更多的关注在过去十.然而, 由于测量成本相对昂贵, 因此它的实际利用可能受到限制, 在这种情况下, 用于细胞分析仪的昂贵的一次性黄金微晶片将被使用。在本议定书中, 我们制定了一个通用的战略, 以再生阵列黄金电极装备为一个商业无标签细胞分析仪。再生过程包括胰蛋白酶消化, 用乙醇和水冲洗, 以及纺纱步骤。该方法已经过测试, 证明对商业电子板的再生和重复使用至少三次是有效的, 这将有助于研究人员节省实时细胞化验的高运行成本。

金微芯片的表面特性: 在图 1中概述了此协议中使用的再生过程。 图 2显示了光学显微镜下的新鲜和再生电子版的显微表面图片。如图 2A和图 2C所示, 显微观察表明, 在新鲜和经过处理的电子板之间, 微晶片表面的光学特性几乎没有差别。使用阿霉素自发荧光?...

Watch this Scientific Journal Video about Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer at JoVE.com

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

该协议描述了一个通用的战略, 以重新生成商业阵列金电极装备的无标签的细胞分析仪, 旨在节省高运行成本 ofmicrochip 的化验。再生过程包括胰蛋白酶消化, 用乙醇和水冲洗, 和一个纺纱步骤, 使微晶片的重复使用。

用于实时细胞分析仪的阵列金电极再生

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

regeneration-arrayed-gold-microelectrodes-equipped-for-real-time-cell

Research

JoVE Journal

Biochemistry

7.1K 次观看.  Nanjing Medical University. 该协议描述了一个通用的战略, 以重新生成商业阵列金电极装备的无标签的细胞分析仪, 旨在节省高运行成本 ofmicrochip 的化验。再生过程包括胰蛋白酶消化, 用乙醇和水冲洗, 和一个纺纱步骤, 使微晶片的重复使用。

视频: Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

高度敏感和快速荧光检测用便携式分析仪FRET

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

科研

生物化学

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

通过智能手机实时跟踪DNA片段分离

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

rad51 介导的 dna 链交换反应的实时观测

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

实现蛋白质快速光化学氧化的实时补偿，用于确定蛋白质地形变化

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...