德国联邦教育和研究部（FKZ 0312138A和FKZ 316159），梅克伦堡 - 西波美拉尼亚与欧盟结构基金（ESF / IVWM-B34-0030 / 10和ESF / IVBM-B35-0010 / 12），DFG（DA1296-1）和德国心脏基金会（F / 01/12）。此外，RD由罗斯托克大学医学中心（889001），DAMP基金会和BMBF（VIP + 00240）的FORUN计划支持。

根据罗斯托克大学医学中心动物护理的道德准则进行了涉及新生小鼠的本议定书中的所有步骤。 1.细胞培养皿和心肌细胞培养基的制备<ol><li>用PBS中的0.1％明胶包被细胞培养板，并在37℃下孵育4小时或在4℃下孵育过夜。取出明胶，使其在无菌层流空气流下干燥。 </li><li>准备由补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的50mL的DMEM组成的细胞培养基。预热至37°C。 </li></ol> 2.新生儿心肌细胞的分离<ol><li>用无菌剪刀斩首新生儿小鼠（1-2天），沿胸骨打开胸部。使用镊子去除心脏，同时轻轻地将胸部按压在一起，并将心脏转移到含有冰冷HBSS的24孔板中（不含Ca 2+和Mg 2+ ）。 </li><li>使用无菌镊子去除非心脏组织和较大的血管。将清洁的心脏转移到含有冰冷HBSS的1.5mL管中。每管最多使用5颗心进行酶消化。 </li><li>用小剪刀将心脏夹碎成0.5至1毫米3片。 </li><li>使用1 mL微量移液管抽吸/添加HBSS，用HBSS洗涤切碎的心脏两次。加入酶前，彻底清除HBSS。 </li><li>对于新生儿心脏的酶消化，使用市售的试剂盒，并遵循制造商的方案（参见材料表 ）。每5分钟摇动含有切碎的心脏的管，同时用酶在37℃下孵育35分钟。 </li><li>将含有细胞培养基的未包被的细胞培养皿上的悬浮细胞悬浮1.5-2小时以允许非心肌细胞部分的粘附。如果高纯度重复此步骤需要心肌细胞。如果需要，进一步培养非心肌细胞部分。 注意:对于15个心脏的细胞悬浮液，可以在75cm 2的细胞培养瓶上进行预镀层步骤。通常，从一个新生儿心脏获得〜5×10 5个细胞。 </li><li>将上清液收集在15 mL锥形管中，并以300 x g离心10 min。将细胞重悬于1mL细胞培养基中。 </li><li>用Neubauer室血细胞计数器计数细胞，或使用等效方法。 </li><li>在密度为3×10 5个细胞/ cm 2的6孔板上平板分离的心肌细胞。在37℃和5％CO 2下孵育细胞过夜。 注意:任选地，细胞也可以在这一点被转染。然而，当在进行电穿孔之前将细胞培养一天时，观察到增加的活力。 </li></ol> 3.用荧光标记的miRNA进行转染<ol><li>预温水细胞培养基（参见步骤1.2）至37℃在水浴或等效装置中。 </li><li>在无RNase的无菌水中制备20μM的荧光miRNA储备溶液。 </li><li>制备含有90mM Na 2 HPO 4，90mM NaH 2 PO 4，5mM KCl，10mM MgCl 2和10mM琥珀酸钠的电穿孔缓冲液，并将pH调节至7.2;电穿孔缓冲液可以在-20°C储存几个月。 </li><li>使用0.05％胰蛋白酶从培养皿中分离细胞5分钟。通过添加细胞培养基灭活胰蛋白酶。 </li><li>用Neubauer室血细胞计数器计数细胞，或使用等效方法。将细胞以300xg离心10分钟。 </li><li>将细胞重悬于电穿孔缓冲液中以获得每100μL4×10 5个细胞的浓度。 </li><li> 将100μL细胞悬浮液与荧光miRNA（终浓度:0.25μM）在管中混合将混合物装入电穿孔试管。 <ol><li>根据细胞类型经验性地确定适当的miRNA数量。 </li></ol></li><li>使用"G-009"程序，使用电穿孔装置进行电穿孔（参见材料表 ）。 </li><li>加入500μL预热后的细胞培养液，将全细胞悬浮液（4×10 5个细胞）转移到4孔玻璃底部载玻片的孔中。在37℃，5％CO 2气氛中培养细胞1天。 注意:对于FRAP分析，细胞密度为〜80％是最佳的。标记的miRNA的转染应该仅使用电穿孔进行。由于标记的miRNA分子的均匀分布对于FRAP测量是有益的，因此不推荐基于试剂的转染。 </li></ol> 4.应用3D-FRAP显微镜（第3天） <ol><li>将显微镜孵化器预热至37°; C，并在FRAP测量前至少2小时开启共焦显微镜系统，建立热平衡并降低漂移的可能性。如果可能，保持5％二氧化碳气氛。 </li><li>将腔室载玻片插入平台样品架。 </li><li>使用1.4 NA油物镜（400倍放大）和561nm激光激发光在低激光功率下寻找转染的心肌细胞簇，检测范围为570-680nm。 注意:使用显微镜专用软件（参见"材料表" ）对FRAP设置，图像采集和分析进行了定义 。 </li><li> 激活"安装管理器"中的"z-Stack"，"时间序列"，"漂白"和"区域"按钮。 </li><li> 定义FRAP参数。 <ol><li>通过设置"帧大小"来定义"采集模式"菜单中的图像采集设置4;到512×512，"行步"为1，"扫描时间"为&lt;1秒。 </li><li>在"通道"菜单中，调整激光功率，偏移和增益设置，以从最小激光激发（ 例如，激光功率:1-5％）获得最大荧光;调整以避免强度饱和。将"针孔尺寸"设置为2μm。 </li><li>接下来，在"区域"菜单中，选择"ROI绘图工具"，并使用光标标记目标单元格，参考单元格和背景区域。如果需要，选择几个目标细胞进行光漂白。 </li><li>调整"漂白"菜单中的漂白设置（ 例如，迭代:9-14，光漂白激光功率:100％，图像采集间隔:60秒，周期:15）。定义3次初始扫描后的漂白开始。 </li><li>在"z-Stack"菜单中，根据单元格的厚度定义z-stack采集的限制。调整"数字of z层"至12 - 15。 </li><li>开始FRAP实验并记录荧光恢复。 注意:由于FRAP实验的设置取决于细胞类型，荧光染料和显微镜系统，最好进行试验性实验以确定FRAP的最佳参数。漂白应足以将初始荧光强度降低至少50％。通常，每30-60秒应记录荧光恢复，直到达到平台期。 </li></ol></li></ol>数据分析<ol><li> 创建所获得的z-叠层的最大投影并获得漂白的靶细胞，参考细胞和背景的荧光强度值。使用显微镜专用软件，点击"处理"→"最大强度投影"→选择文件→"应用"。 <ol><li>或者，使用等效图像分析工具（ <e例如，ImageJ）。 </li></ol></li><li> 将所有时间点的荧光强度数据从目标单元格，背景和参考单元复制到电子表格中。 <ol><li>从目标单元的强度值中减去参考单元的背景强度和强度，以校正在采集过程中产生的漂白。对每个时间点执行背景和参考单元校正。 </li><li>通过将每个值除以初始荧光，将校正的FRAP数据归一化为漂白前的初始荧光强度。 </li><li>为了获得FRAP曲线，通过从每个时间点的强度值中减去，将基线设置为漂白后立即的荧光强度。 </li></ol></li></ol>

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Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery+of+miRNAs+for+cancer+therapy:+Challenges+and+strategies.">In vivo delivery of miRNAs for cancer therapy: Challenges and strategies.</a> Adv Drug Deliv Rev. 81C, 128-141 (2015).</li></ol>

作者宣称没有利益冲突。

<html> miRNA是细胞生理学中的关键参与者，并被证明通过使用G_s作为细胞间交换途径，作为信号分子11,12,22。目前的方案提出体外活细胞成像技术来表征使用荧光miRNA在细胞簇内的GJ依赖性穿梭。 该方案作为细胞模型系统在心肌细胞上开发。然而，如果电穿孔是miRNA转染的合适方法，则该方法可以应用于几种细胞类型。除了细胞间细胞交换，所提出的技术也适用于研究miRNA?...

小的非编码RNA是细胞基因调控的重要参与者。这些分子由与特异性靶mRNA结合的20-25个核苷酸组成，导致翻译的阻断或mRNA降解1,2 。由小RNA（例如miRNA和siRNA）进行的基因调控过程是许多不同物种中发现的高度保守的机制。特别地，miRNA分子对多种生理过程至关重要，包括增殖，分化和再生4,5 。此外，miRNA表达的失调归因于许多病理学障碍。相应地，miRNA已经被证明适合作为诊断的生物标志物和用作基因治疗的治疗剂6,7 </suP&gt;。 间隙连接（GJs）是两个相邻细胞的质膜中的专门的蛋白质结构，允许分子量高达1kD的扩散交换。已被证明对于组织发育，分化，细胞死亡和病理性疾病如癌症或心血管疾病8,9,10很重要 。已经描述了几个分子能够穿过GJ通道，包括离子，代谢物和核苷酸。有趣的是，还发现GJs为小RNA 11,12的细胞间移动提供了途径。因此，miRNA不仅可以在其产生的细胞内，而且在受体细胞内起作用。这突出了miRNA在细胞间信号转导系统中的作用。同时，数据显示该间隙连接细胞间通讯与miRNA功能密切相关。由于miRNA和GJs对组织稳态，病理学和诊断的重大影响，全面了解GJs的功能和miRNA的相关细胞间动力学有助于阐明基于miRNA的疾病的机制，并制定新的策略miRNA疗法。 根据间隙连接耦合的程度，miRNA分子在细胞间的转移可以是非常快速的过程。因此，需要一种允许可视化和量化这些调节信号分子的快速细胞间移动的方法。通常，已经应用流式细胞术和分子生物学技术来证明小RNA的穿梭11,12,13,14。然而，与FRAP微观相反复制，这些方法缺乏高时间分辨率，这是通过GJs分析miRNA的交换时强制的。此外，FRAP显微镜侵入性较小，因此代表了一种功能强大且新颖的活细胞成像技术，用于评估几种15,16,17型细胞中分子的GJ依赖性交换。 在这里，我们提出了一个详细的协议，描述了3D-FRAP应用于评估心肌细胞之间的miRNA穿梭。为此，用荧光标记的miRNA转染心肌细胞。用该miRNA标记的细胞被光漂白，并且以时间依赖的方式记录来自相邻细胞的间隙连接miRNA再流入。 FRAP实验的高时间分辨率提供了进行动力学研究以准确评估活细胞之间的miRNA和siRNA的细胞间转移的可能性。 Moreo由于小RNA可以通过具有高度不同动力学的不同机制进行交换，FRAP显微镜可以帮助澄清GJ在相应的穿梭过程18中涉及的程度。此外，3D-FRAP可用于研究GJ通透性的生理和病理学改变及其对小RNA转移的影响15,19。

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">neonatal NMRI mice</td>
			<td style="width:159px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Gelatin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">G7041</td>
			<td style="width:179px;">0.1% solution in PBS, sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PBS</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-53500</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Dulbecco&#180;s modified medium</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-03550</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin/Streptomycin</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140122</td>
			<td style="width:179px;">100 U/ml, 100&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal bovine serum</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P30-3306</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Cell culture plastic</td>
			<td style="width:159px;">TPP</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Primary Cardiomyocyte Isolation Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">HBSS</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;">included in primary cardiomyocyte isolation kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypan blue</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">miRIDIAN Dy547 labeled microRNA Mimic&#160;</td>
			<td style="width:159px;">Dharmacon</td>
			<td style="width:119px;">CP-004500-01-05</td>
			<td style="width:179px;">dissolve in RNAse free water, stock solution 20&#181;M</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:243px;">Sodium phosphate monobasic</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">S3139</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium phosphate dibasic</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">T877.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium chloride</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Magnesium chloride</td>
			<td style="width:159px;">Serva</td>
			<td style="width:119px;">28305.01</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium succinate</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">3195.1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin/EDTA solution</td>
			<td style="width:159px;">Merck</td>
			<td style="width:119px;">L2153</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">4-well- Glass bottom chamber slides</td>
			<td style="width:159px;">IBIDI</td>
			<td style="width:119px;">80827</td>
			<td style="width:179px;">coat with 0.1% gelatin solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Amaxa Nucleofector II</td>
			<td style="width:159px;">Lonza</td>
			<td style="width:119px;"></td>
			<td style="width:179px;">program G-009 was used for Electroporation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LSM 780 ELYRA PS.1 system</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Excel software</td>
			<td style="width:159px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#945;-actinin antibody</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:119px;">ab9465</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Connexin43 antibody</td>
			<td style="width:159px;">Santa Cruz</td>
			<td style="width:119px;">sc-9059</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-mouse Alexa 594 antibody</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11005</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-rabbit Alexa 488 antibody&#160;</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11034</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Connexin43 siRNA</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM16708 ID158724</td>
			<td>&#160;final concentration 250nM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Near-IR Live/Dead Cell Stain Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CellTrace Calcein Red-Orange</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DAPI nuclear stain</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy

小反义RNA（如miRNA和siRNA）在细胞生理学和病理学中起重要作用，而且可用作治疗几种疾病的治疗剂。开发miRNA / siRNA治疗新的创新策略是基于对基础机制的广泛了解。最近的数据表明，小细胞以间隙连接依赖的方式在细胞之间进行交换，从而在受体细胞中诱导基因调控作用。分子生物学技术和流式细胞仪分析通常用于研究miRNA的细胞间交换。然而，这些方法不提供高的时间分辨率，这在研究分子的间隙连接通量时是必需的。因此，为了研究miRNA / siRNA作为细胞间信号分子的影响，需要新的工具，这将允许在细胞水平上分析这些小RNA。本协议描述了ap光漂白后三维荧光恢复（3D-FRAP）显微镜的澄清，以阐明心脏细胞之间的miRNA分子的间隙连接依赖性交换。重要的是，这种直接和非侵入性的活细胞成像方法允许实时荧光标记的小RNA的间隙连接穿梭的可视化和定量，具有高的时空分辨率。通过3D-FRAP获得的数据证实了细胞间基因调控的新途径，其中小RNA作为细胞间网络内的信号分子。

在这里，我们提出3D-FRAP显微镜作为非侵入性技术研究新生儿心肌细胞荧光miRNA间隙连接穿梭。分离的心肌细胞显示典型的条纹α-肌动蛋白模式，并且沿着细胞 - 细胞边界包含大量的Cx43斑块（ 图1A ，白色箭头），这允许分子的高细胞间通量。通过显微定量α-肌动蛋白阳性细胞评估分离的心肌细胞的纯度。虽然培养物中存在一些非心肌细胞（α-肌动蛋?...

Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

在这里，我们描述了光漂白后三维荧光恢复（3D-FRAP）的应用，用于分析miRNA的间隙连接依赖穿梭。与通常应用的方法相比，3D-FRAP允许实时定量小RNA的细胞间转移，具有高的时空分辨率。

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

analysis-gap-junction-dependent-transfer-mirna-with-3d-frap

Research

JoVE Journal

Biology

8.8K 次观看.  Reference and Translation Center for Cardiac Stem Cell Therapy (RTC). 在这里，我们描述了光漂白后三维荧光恢复（3D-FRAP）的应用，用于分析miRNA的间隙连接依赖穿梭。与通常应用的方法相比，3D-FRAP允许实时定量小RNA的细胞间转移，具有高的时空分辨率。 

视频: Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

科研

生物学

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

自噬的分析<em&gt;产黄青霉</em&gt;通过结合使用饥饿垫荧光显微镜

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

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 ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

系绳双层脂膜，以监测金纳米粒子和脂膜之间的热传递

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

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