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 中使用的阵列金微晶片的表面性能造成极大的危害。因此, 一种温和有效的方法是提出的再生策略发展的主要考虑因素。其他同样重要的因素是环境问题, 因为这些一次性微晶片经常与靶向癌细胞一起使用。
在本协议中, 我们介绍了一种用于 RTCA 的商用阵列金微晶片再生的通用方法。我们的协议是建立在胰蛋白酶消化和漂洗与乙醇和水。额外的纺纱步骤被认为是有效的去除不溶性颗粒用于再生过程的化验。与以前报告的方法中使用的试剂相比, 再生试剂胰蛋白酶和乙醇可随时供日常实验室使用, 这些生物产品/化学品毒性低, 因此被认为是环境友好, 这是可取的目的, 再生。
在该协议中, 关键步骤是胰蛋白酶消化, 以确保金微晶片的再生性能。trypsinization 的优化, 包括浓度和时间, 是实现再生的首要任务。除了在我们以前的报告19中合理化的消化时间外, 对优化过程的一个很好的评估是简单地使用光学显微镜检查表面特性。
另一个问题是在再生过程中处理微晶片。由于在实验中经常使用吸管, 因此应注意避免在微芯片表面划伤尖端。此外, 它有利于预先消毒的提示, 纱布, 勺子, 以确保再生效率。最后但并非最不重要的是, 再生过程应在生物安全罩中进行, 以避免任何污染。
应该指出, 再生协议中涉及的冲洗过程是手工进行的, 这必然会导致不同人员的偏差。为了克服这一限制, 自动或半自动设备的冲洗目的可以定制设计, 考虑到模块化的电子板使用。此外, 已对再生协议进行了测试, 以有效地进行基于细胞的化验。然而, 在分子相互作用很强的情况下, 如芯片界面上的抗体抗原形成, 所提出的再生条件可能在很大程度上受到限制, 应考虑相对较强的缓冲条件。
总的来说, 目前的再生协议已经测试, 以允许重复使用商业金微晶片, 以及其他导电电极, 如 Au 和玻碳电极。我们预计, 该协议的应用将节省的高运行成本的实时芯片的细胞化验。从长远来看, 基于微芯片的检测越来越多地应用于蛋白质组学和细胞分析, 本研究提出的协议可以为再生提供一个选择, 即自动或半自动冲洗设备可以在累积的一次性芯片的情况下设计和应用。

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><tbody><tr><td>Rat: Sprague-Dawley</td><td>Charles River</td><td>Cat# 400</td><td></td></tr><tr><td>mouse anti-rat CD11b monoclonal (clone OX42)</td><td>Bio-Rad</td><td>Cat# MCA275R</td><td>Panning: 1:1,000; Staining: 1:500</td></tr><tr><td>Goat polycolonal anti-Iba1&amp;nbsp;</td><td>Abcam</td><td>Cat# AB5076</td><td>Staining: 1:500</td></tr><tr><td>Rabbit polyclonal anti-Ki67&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>Cat# AB15580</td><td>Staining: 1:500</td></tr><tr><td>Alexa Fluor Donkey anti-mouse 594</td><td>Invitrogen</td><td>Cat# 11055</td><td>Staining: 1:500</td></tr><tr><td>Alexa Fluor Donkey anti-goat 488</td><td>Invitrogen</td><td>Cat# A-21203</td><td>Staining: 1:500</td></tr><tr><td>Alexa Fluor Donkey anti-Rabbit 647</td><td>Invitrogen</td><td>Cat# A-31573</td><td>Staining: 1:500</td></tr><tr><td>Triton-X (detergent in ICC staining)</td><td>Thermo Fisher</td><td>Cat# 28313</td><td></td></tr><tr><td>Heparan sulfate</td><td>Galen Laboratory Supplies</td><td>Cat# GAG-HS01</td><td></td></tr><tr><td>Heparin</td><td>Sigma&amp;nbsp;</td><td>Cat# M3149</td><td></td></tr><tr><td>Peptone from milk solids</td><td>Sigma</td><td>Cat# P6838</td><td></td></tr><tr><td>TGF-&amp;beta;2</td><td>Peprotech</td><td>Cat# 100-35B</td><td></td></tr><tr><td>Murine IL-34</td><td>R&amp;amp;D Systems</td><td>Cat# 5195-ML/CF</td><td></td></tr><tr><td>Ovine wool cholesterol</td><td>Avanti Polar Lipids</td><td>Cat# 700000P</td><td></td></tr><tr><td>Collagen IV</td><td>Corning&amp;nbsp;</td><td>Cat# 354233</td><td></td></tr><tr><td>Oleic acid</td><td>Cayman Chemicals&amp;nbsp;</td><td>Cat# 90260</td><td></td></tr><tr><td>11(Z)Eicosadienoic (Gondoic) Acid</td><td>Cayman Chemicals&amp;nbsp;</td><td>Cat# 20606</td><td></td></tr><tr><td>Calcein AM dye</td><td>Invitrogen</td><td>Cat# C3100MP</td><td></td></tr><tr><td>Ethidium homodimer-1</td><td>Invitrogen</td><td>Cat# E1169</td><td></td></tr><tr><td>DNaseI</td><td>Worthington</td><td>Cat# DPRFS</td><td></td></tr><tr><td>Percoll PLUS</td><td>GE Healthcare</td><td>Cat# 17-5445-02</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Sigma</td><td>Cat# T9935</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>Cat# 21041-02</td><td></td></tr><tr><td>Penicillin/ Streptomycin</td><td>Gibco</td><td>Cat# 15140-122</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>Cat# 25030-081</td><td></td></tr><tr><td>N-acetyl cysteine&amp;nbsp;</td><td>Sigma</td><td>Cat# A9165</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>Cat# 16634</td><td></td></tr><tr><td>Apo-transferrin&amp;nbsp;</td><td>Sigma</td><td>Cat# T1147</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma</td><td>Cat# S-5261</td><td></td></tr><tr><td>DMEM (high glucose)</td><td>Gibco</td><td>Cat# 11960-044</td><td></td></tr><tr><td>Dapi Fluoromount-G</td><td>Southern Biotech</td><td>Cat# 0100-20&amp;nbsp;</td><td></td></tr><tr><td>Poly-D-Lysine&amp;nbsp;</td><td>Sigma</td><td>Cat# A-003-E</td><td></td></tr><tr><td>Primaria Plates&amp;nbsp;</td><td>VWR</td><td>Cat# 62406-456</td><td></td></tr><tr><td>&lt;strong&gt;Stock reagents&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;reconstitution&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Concentration used&lt;/strong&gt;</td><td>&lt;strong&gt;Storage&lt;/strong&gt;</td></tr><tr><td>Apo-transferrin</td><td>10 mg/mL in PBS&amp;nbsp;</td><td>1:100</td><td>-20&amp;deg;C</td></tr><tr><td>N-acetyl cysteine</td><td>5 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>1:1,000</td><td>-20&amp;deg;C</td></tr><tr><td>Sodium selenite</td><td>2.5 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>1:25,000</td><td>-20&amp;deg;C</td></tr><tr><td>Collagen IV</td><td>200 &amp;mu;g/mL in PBS&amp;nbsp;</td><td>1:100</td><td>-80&amp;deg;C</td></tr><tr><td>TGF-b2</td><td>20 &amp;mu;g/mL in PBS&amp;nbsp;</td><td>1:10,000</td><td>-20&amp;deg;C</td></tr><tr><td>IL-34</td><td>100 &amp;mu;g/mL in PBS&amp;nbsp;</td><td>1:1,000</td><td>-80&amp;deg;C</td></tr><tr><td>Ovine wool cholesterol</td><td>1.5 mg/mL in 100% ethanol&amp;nbsp;</td><td>1:1,000</td><td>-20&amp;deg;C</td></tr><tr><td>Heparan sulfate</td><td>1 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>1:1,000</td><td>-20&amp;deg;C</td></tr><tr><td>Oleic acid/Gondoic acid</td><td>Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol&amp;nbsp;</td><td>1:1,000</td><td>-20&amp;deg;C</td></tr><tr><td>Heparin</td><td>50 mg/mL in PBS&amp;nbsp;</td><td>1:100</td><td>-20&amp;deg;C</td></tr><tr><td>&lt;strong&gt;Solutions&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Recipe&amp;nbsp;&lt;/strong&gt;</td><td></td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Perfusion Buffer</td><td>50 &amp;mu;g/mL heparin in DPBS&lt;sup&gt;++&lt;/sup&gt; (PBS with Ca&lt;sup&gt;++&lt;/sup&gt; and Mg&lt;sup&gt;+ +&lt;/sup&gt;)</td><td></td><td>Use when ice-cold</td></tr><tr><td>Douncing Buffer</td><td>200 &amp;mu;L of 0.4% DNaseI in 50 mL of DPBS&lt;sup&gt;++&lt;/sup&gt;</td><td></td><td>Use when ice-cold</td></tr><tr><td>Panning Buffer</td><td>2 mg/mL of peptone from milk solids in DPBS&lt;sup&gt;++&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>Microglia Growth Medium (MGM)</td><td>DMEM/F12 containing 100 units/mL penicillin, 100 &amp;mu;g/mL streptomycin, 2 mM glutamine, 5 &amp;mu;g/mL N-acetyl cysteine, 5 &amp;mu;g/mL insulin, 100 &amp;mu;g/mL apo-transferrin, and 100 ng/mL sodium selenite</td><td></td><td>Use ice-cold MGM to pan microglia off of immnopanning dish.</td></tr><tr><td>Collagen IV Coating</td><td>MGM containing 2 &amp;mu;g/mL collagen IV</td><td></td><td></td></tr><tr><td>Myelin Seperation Buffer</td><td>9 mL Percoll PLUS, 1 mL 10x PBS without Ca&lt;sup&gt;++&lt;/sup&gt; and Mg&lt;sup&gt;++&lt;/sup&gt;, 9 &amp;mu;L 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 5 &amp;mu;L 1 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td>Mix well after the addition of&amp;nbsp; CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>TGF-b2/IL-34/Cholesterol containing growth media (TIC)&amp;nbsp;</td><td>MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &amp;mu;g/mL ovine wool cholesterol, 10 &amp;mu;g/mL heparan sulfate, 0.1 &amp;mu;g/ml oleic acid, and 0.001 &amp;mu;g/ml gondoic acid</td><td></td><td>Make sure to add cholesterol to media warmed to 37 &amp;deg;C and do not add more than 1.5 &amp;mu;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO&lt;sub&gt;2&lt;/sub&gt; for 30 min- 1 hr before adding to cells to insure optimal pH.&amp;nbsp;</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的细胞, 以方便评估细胞状态, 我们观察到在再生后金微晶片上培养细胞的相似同质性 (图 2B和图2D).此外, 孵化的癌细胞显示了一个明确定义的细胞形态学的电子板块表明芯片的可重用性。图 3显示了新鲜和再生电子板的拉曼光谱。在新鲜和被处理的电子板材之间的几乎相同的拉曼峰值分布表明金微晶片表面的再生过程是成功的。A549 细胞覆盖的电子板的拉曼信号在一定程度上被扭曲, 可能是由于 A549 细胞自发荧光的干扰引起的。
使用 RTCA 的细胞毒性试验再生微晶片:我们还应用了再生协议的细胞增殖和物质细胞毒性评估的测试 (图 4)。将最佳密度的 A549 细胞播种到再生电子板16上, 用实时细胞分析仪记录生长动力学曲线。如图 4A所示, 平行实验井产生的所有生长曲线均具有足够的一致性, 这表明在再生后, 电子板16的表面电状态很大程度上保持不变。利用介孔二氧化硅材料对再生电子板 L8 进行了细胞毒性试验, 如图 4B所示。与对照试验相比, 介孔材料处理样品的 CI 在一定程度上有所下降, 表明材料的微细胞毒性。介孔材料对 Dox 抗癌药物的进一步封装和释放, 导致 A549 生长曲线显著下降。这些实验结果表明, 使用新的电子板和以前报告的工作19的一致性, 验证了此建议的再生协议的有效性。
电化学方法评估的再生效率:以商用金电极和玻碳电极为试验平台, 对再生效率进行了评价。图 5显示了用癌细胞修饰的 Au 电极和剑桥的 EIS。结果表明, A549 细胞在电极上孵化后, 与裸电极相比, 明显增加了电子转移电阻 (Ret)。在金电极和剑桥经处理后, 根据图 5中的协议对 Ret 进行了显著降低。金电极和通用电气的相同变化表明, 微芯片对其表面电性能有一个有效的、普遍的再生过程。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56250/56250fig1.jpg"> 图 1: 电子版的再生过程.在典型的再生运行中, 四主要步骤包括孵化细胞以及它们在电子板表面的脱离, 随后是漂洗、纺纱和紫外线灭菌步骤。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56250/56250fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56250/56250fig2.jpg"> 图 2: 电子板的表面特性.(A)新的电子版(B) A549 细胞被镀在. (C)再生电子板(D) A549 细胞被镀在 C 上。对于附着在金微晶片上的细胞, 使用了 DOX。图 2A和图 2C已从徐、Z.等。19<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56250/56250fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56250/56250fig3.jpg"> 图 3: 不同处理过的电子板的拉曼光谱.(A)新的电子版。(B)新的 A549 细胞种子电子板。(C)再生电子版。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56250/56250fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56250/56250fig4.jpg"> 图 4: 在三次再生电子板上 A549 细胞生长曲线.(A)在电子板上播种的细胞的平行生长曲线16。(B)对每种孵化井的记录曲线的误差条 (标准偏差、STD) L8 电子板上的介孔二氧化硅材料的细胞毒性试验。图 4A已从徐、Z. et . 中修改。19<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56250/56250fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56250/56250fig5.jpg"> 图 5: 不同修饰金电极和玻碳电极在10毫米氯化钾电解质溶液中的电化学阻抗谱, 含1毫米 K3[Fe (CN)6]。(A)不同处理的金电极。(B)不同的经处理的普通教育。图 5A已被修改 fromXu, Z. et 。19<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56250/56250fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>芯片应用</td>
			<td>再生缓冲器</td>
			<td>再生策略</td>
		</tr>
<tr>
<td>表面等离子体共振 (SPR)</td>
			<td>食人鱼解决方案 (浓缩 H2所以4/30% H2O2 = 3:1, v/v)17
</td>
			<td>基于强氧化的分析和探针剥离</td>
		</tr>
<tr>
<td rowspan="2">压电悬臂梁传感器 (PEMS)</td>
			<td>甘氨酸-HCl 混合物23,24
</td>
			<td rowspan="2">分离目标探针和去除分析物</td>
		</tr>
<tr>
<td>高盐浓度溶液 (2M 氯化镁2 + 1.5 M 三)25
</td>
		</tr>
<tr>
<td>金膜</td>
			<td>50mM KOH+25%h2O226
</td>
			<td>氧化有机物</td>
		</tr>
</tbody></table>表 1: 报告的再生战略的简要摘要。

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

Nanyang Technological University

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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

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

科研

生物工程

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

对于系统电化学神经记录电极和电生理评价方法

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

神经科学

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

体外 通过微有机电荷调制场效应晶体管阵列进行多参数蜂窝分析

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This paper presents a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a Parylene C deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. This fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. 
The three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1 kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows that UV laser-cut fibers can record neural signals in vivo. Existing carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. This carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

开源工具包:用于神经记录的台式碳纤维微电极阵列

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

机械节点孔传感:用于多参数单细胞粘弹性测量的快速、无标记平台

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

用于慢性和急性体内记录的碳纤维微电极阵列 的 构建和实现

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...

Electrophysiological Recordings For Assessing Neuronal Regenerations in Co-cultured Spinal Cord Slices

Put a Petri dish with a coated microelectrode array inside under a stereomicroscope. Bring the array into focus, and center a six-microliter droplet of chicken plasma on the electrode array. Using a small spatula, carefully slide two spinal cord sections with their ventral sides facing each other into the plasma droplet.
Next, add eight microliters of thrombin around the chicken plasma droplet. Then, use the pipette tip to carefully mix and spread the chicken plasma and thrombin mixture. Just before the chicken plasma and thrombin mixture becomes too stringy and starts to coagulate, aspirate the excess liquid. Then, cap the Petri dish and place it in a humidified chamber.
Place the chamber inside an incubator at 37 degrees Celsius for about an hour. After incubation, carefully add 10 microliters of nutrient medium to the sample. Cap the Petri dish and place it back into the incubator for an additional 45 minutes. Next, place each of the multi-electrode array culture assemblies into a roller tube and add three milliliters of nutrient medium. Close the lid tightly and place the roller tube into the roller drum. Rotate the drum at 1 to 2 RPM in the incubator at 37 degrees Celsius.
Using sterile rubber-tipped forceps, remove the multi-electrode array culture assemblies from the roller tube and place them into a Petri dish under a stereomicroscope. Bring the tissue into focus and verify that the two slices are fused. Next, hold the assembly steady and place a scalpel blade in the groove of the multielectrode array close to the tissue slices. Hold the scalpel rather horizontally and then lift the scalpel handle up, but let the scalpel blade stay in the groove of the array in such a way that the blade rolls from base to tip, cutting through the tissue, covering the groove.
Sever any residual tissue connections with a 25-gauge needle tip if necessary. Work only in the area within the groove, and do not touch the tender edges. Put the multi-electrode array culture assemblies back into the roller tube and add three milliliters of fresh nutrient medium to the cultures. Then, place the roller tube back onto the roller drum in the incubator at 37 degrees Celsius.
Mount a multi-electrode array culture assembly into a recording chamber and apply about 500 microliters of extracellular solution to the array. Then, mount the assembly on the microscope. Wait 10 minutes for the system to stabilize, and then, record basic spontaneous activity from each of the activity-detecting electrodes for 10 minutes. Repeat the recordings a total of two times.
To ensure stable extracellular conditions, exchange the extracellular solution after every recording session. If desired, disinhibit the network by applying extracellular solution containing one micromolar of strychnine and 10 micromolar of gabazine and wait for at least two minutes before recording the electrical activity.

用于评估共培养脊髓切片中神经元再生的电生理记录

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

EoE Sub Articles

Constructing Patterned Neuronal Circuits on a Multi-Electrode Array

First, clean the multielectrode array by washing it under tap water, and then sonicating it in a concentrated enzymatic detergent. Repeat these steps three times. Then, sonicate the MEA in pure distilled water three times. After which, under a hood, wash the MEA with distilled water before sterilizing it with UV light for 30 minutes.
Now, attach a stencil to a prepared micromanipulator. Under an inverted microscope, inspect and align the patterned structure to match the electrodes of the MEA. Then, use the micromanipulator to lower the stencil to the MEA surface. Once attached, raise the micromanipulator. Now, transfer the MEA to the vacuum chamber for 15 minutes. Then, apply a 1-milliliter drop of PDL to the stencil, and return it to the vacuum chamber for 2 cycles of 20 minutes.
Let the PDL dry overnight. The next day, remove the PDMS stencils from the MEAs using tweezers. Lastly, UV-sterilize the MEAs for 7 minutes. They are then ready for the cells. In preparation, culture cells and prepare suspensions as in the text protocol. After resuspending the required number of cells, plate them at the center of the patterned area on the MEA or coverslip, and MEA should be loaded with 100-microliter volumes, and a coverslip accommodates 1,000 microliters. Incubate the plated cells for 40 minutes. Then, add plating medium.
To maintain the cells every 4 days, add back fresh supplemented growth medium. After the sixth or seventh days, neuronal connections between the modules should start forming.

用于实时细胞分析仪的阵列金电极再生

摘要

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此视频中的章节

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

对于系统电化学神经记录电极和电生理评价方法

神经活动的传播的展开海马准备具有穿透微电极阵列

调查器官脊髓联合培养物中生长多电极阵列功能再生

体外通过微有机电荷调制场效应晶体管阵列进行多参数蜂窝分析

开源工具包:用于神经记录的台式碳纤维微电极阵列

机械节点孔传感:用于多参数单细胞粘弹性测量的快速、无标记平台

用于慢性和急性体内记录的碳纤维微电极阵列的构建和实现

用于评估共培养脊髓切片中神经元再生的电生理记录

在多电极阵列上构建图案化神经元回路

用于实时细胞分析仪的阵列金电极再生

摘要

探索更多视频

此视频中的章节

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

对于系统电化学神经记录电极和电生理评价方法

神经活动的传播的展开海马准备具有穿透微电极阵列

调查器官脊髓联合培养物中生长多电极阵列功能再生

体外 通过微有机电荷调制场效应晶体管阵列进行多参数蜂窝分析

开源工具包:用于神经记录的台式碳纤维微电极阵列

机械节点孔传感:用于多参数单细胞粘弹性测量的快速、无标记平台

用于慢性和急性体内记录的碳纤维微电极阵列 的 构建和实现

用于评估共培养脊髓切片中神经元再生的电生理记录

在多电极阵列上构建图案化神经元回路

体外通过微有机电荷调制场效应晶体管阵列进行多参数蜂窝分析

用于慢性和急性体内记录的碳纤维微电极阵列的构建和实现