这项工作是由比利时国家基金资助科学的调查一下（FNRS），校际景点波兰人计划 - 比利时科学政策和“Communauté法语培训中心比利时，操作德RecherchesConcertées”到FC，从以色列科学基金会耶路撒冷（支持ISF），以MM（格兰特＃1311至1312年），以及新墨西哥州（格兰特＃1312至1312年）。

1，准备解决方案<ol><li>制备等渗（600毫渗透摩尔浓度）和低渗含有10mM的KCl，1mM的氯化钙 ，和（500毫渗透摩尔浓度）溶液8 M 2（N-吗啉）-ethanesulphonic磺酸（MES），pH为5.7，并与适当量调节渗透压D-山梨醇：540毫米为等渗和440毫米的低渗溶液。验证的溶液（在目标值的3％）的用渗透压计的摩尔渗透压浓度。 </li><li>制备“酶混合物”含有下列酶的干股：0.55克纤维素，0.1g的果胶酶，0.33克聚乙烯吡咯烷酮K-30，0.33克BSA（见下面的表1），通过涡旋混合的干燥粉末，使5.7毫克等分试样并储存于-20℃。 </li></ol> 2，隔离拟南芥叶肉原生质体<ol><li>准备一个培养皿（10厘米）约6滴（大约各30微升）的等渗溶液。 </li><li>皮尔的背面（下）拟南芥叶表皮，CUT斯达康剥离的叶成约4×4 平方毫米的正方形，然后将正方形的等渗溶液滴用暴露的背面朝下，触摸解决方案。 </li><li>溶解5.7毫克的酶混合物在165微升的1.5毫升管的等渗溶液（3.3％W / W），轻轻混合，通过移液为一分钟左右，直至溶解，并放置几个类似滴酶溶液在相同的Petri这道菜。 </li><li>转叶片到酶溶液滴，收菜的密封盖子有一个圆形封口膜，静置20分钟，浮在水浴设定为28℃的菜。 </li><li>添加几个滴等渗溶液的菜（2滴每各酶溶液滴）。每个叶片转移到一个新的等渗溶液滴，然后，依次向第二降（以洗涤酶溶液之外）。使用产钳提起一块的边缘，摇晃它在第二个下拉（如袋泡茶）以释放原生质体。收集的原生质体（使用剪切掉100微升枪头）滴至1.5 ml管。 </li></ol> 3，低渗挑战分析：拟南芥叶肉细胞肿胀<ol><li>通过填充一个柱用等渗溶液和另一列的低渗溶液中制备的灌注系统（ 图1A）。打开阀门，让一些清水流量（第低渗，那么等渗），以填补管一路下跌到进气歧管（ 图1B）。确保没有被困的气泡，然后关闭阀门。 </li><li>密封盖玻片，用硅脂（ 表1），以使有机玻璃滑动内底的腔室（ 图1B;还看到在图1C的腔室的示意图）。为了使该腔室的底部（盖玻片的向上朝向的暴露表面的油脂环内）的“粘性”的原生质体，大衣呢带正电荷的轴承硫酸鱼精蛋白（1％的水溶液; 表1）或聚-L-赖氨酸（在水中的0.1％， 表1）。传播这种“胶水”在用枪头盖玻片，等待1 - 2分钟，冲洗3 - 4倍的等渗溶液，摇匀掉剩余的溶液。 </li><li>填充腔室向上与等渗溶液。然后，加入一滴含有的解决了室内原生质体，利用剪切掉枪头，等待3 - 4分钟的原生质体沉降。盖上透明盖（ 图1D，1E）室接触到溶液的表面（避免诱捕气泡下方）。 </li><li>将滑动件（轻轻！）上的倒置显微镜台，其连接到灌注系统和泵（防范气泡在管！）和打开在1毫升/分钟的等渗溶液流量为恒定灌注（更快的速率可以使用多达4毫升/分钟）。 </li><li>录音量的变化，倒置显微镜时，用20倍的目标，并有CCD摄像机连接到PC电脑。使用ImageJ的软件的“中央结算系统1394摄像头驱动程序”插件（看具体材料的表，这两个软件作品的下载地址）来记录选择动原生质体的60秒视频电影（大概是，那些粘在底部）在1张/秒（1赫兹）的速率。开始用等渗溶液的15秒冲洗的记录（这构成了基线），切换到45秒的低渗溶液（以完成从灌注的开始共60秒）。保存电影中的TIF格式。注意：选择一个视野与尽可能多的细胞成为可能，满足以下条件：球形，有重点突出的细胞轮廓在其最大周长（ 图2A）。 </li></ol>细胞体积变化使用ImageJ的分析4。 注：分析一系列的膨胀单元的图像，使用“图像浏览器”和“原生质体分析仪插件在ImageJ的软件（写泽维尔Draye）14。与所选择的原生质体在他们的第一个时间点开始，“原生质体分析仪插件会自动检测原生质体的边缘（轮廓），并在实验过程中计算出各自领域的时间过程（该插件可与PfFit分析程序，下同） 。 <ol><li>开始ImageJ的。要打开电影，点击“文件”的ImageJ的面板上，然后，连续的下拉菜单，因为他们展开：“导入”，然后“图像浏览器”。突出显示所选择的电影，然后在其上单击鼠标右键，然后在“原生质体分析仪左键单击。通过电影（用滑块在原生质体图像底部），以确定基本保持不动的在实验过程中的原生质体览 - 这些将被分析。回到第一个IMAGE，使用鼠标，画出围绕选定的原生质体（ 图2B），圆（从ImageJ的绘图工具采摘），然后在“检测参数”中出现的表&#39;确定&#39;。 </li><li>要启动原生质体检测算法，点击“本地”的形象原生质体顶部面板上，然后&#39;过程&#39;在下拉菜单中。检查围绕所选的原生质体（ 图2C）在整个电影的绿色圆圈。保存在Excel文件中的“结果”。退出ImageJ的。注：如果出现一个红点（表示一个坏的轮廓贴合 - 通常是由于一个贫穷的图像对比度），使用不同的参数重新运行。 </li><li>分离属于各小区中的线（其中 - 如果两个或更多的细胞，同时分析 - 将被交织，因为在分析完成一帧一帧），在Excel中，通过细胞数列（“对象”所保存的数据进行排序）。 </li><li>到DETErmine的像素到微米的转换系数获取的P F的实际值，通过相同的20倍显微物镜单元测微尺的图像。拖线（从ImageJ的绘图工具采摘）在标尺的形象和读取相当于标尺长度在ImageJ的主面板底部的像素数。转换在Excel文件中的任意像素面积值到2微米。保存区时程（分别为每个单元）作为一个文本文件（两个数字仅列）。注意：这将是一个输入量拟合&#39;PfFit“计划。 </li></ol> 5，造型渗透压的变化率在实验厅内使用ImageJ和MATLAB程序P F飞度<ol><li>加入2毫克二甲苯蓝（下面的表1）到100毫升等渗溶液（以产生的“指示剂染料”）的。 </li><li>制备灌注系统（如3.1）与指示剂染料和非染色ħypotonic解决方案。 </li><li>密封用硅润滑脂的有机玻璃室的底部盖片，然后轻轻地填充与指示剂染料的腔室，盖上盖玻片（与原生质体之前），并将其放置在显微镜工作台上。 </li><li>该腔室连接到灌注系统和泵，并打开指示剂染料流量在1毫升/分钟的恒定灌注。 </li><li>记录以1 Hz的速率的60秒短片。开始记录用15秒指示剂染料，切换到45秒的低渗溶液。停止拍摄。同花顺的指示剂染料（至少30秒），然后开始一个新的电影。重复约5 - 6次，并保存所有的电影</li><li>使用ImageJ的软件来分析指示剂染料透射率的视频图像，以获得变化的透光率的平均时间过程。 <ol><li> ImageJ的开始，单击“文件”，那么，“打开”，浏览的电影。对于每部电影，画一个10像素宽的垂直矩形影片的第1次图像上的任何地方。点击ImageJ的主面板上的“图像”，然后在下拉菜单中选择“裁剪”。 </li><li>要对齐成一排（共60秒的影片）的60帧，然后再次单击“图像”，然后单击连续在下拉菜单中，他们展开：“栈”和“制作蒙太奇”（列60，行1）。沿着图像整排的任何位置画一个1像素高的横长方形，然后单击“分析”在ImageJ的主面板，然后单击“剧情简介”中的下拉菜单。注：窗口会出现（未示出）&#39;蒙太奇的情节“，而透射率数据的列表可以从菜单中打开。影片的各个图像被表示在这个名单由10透光度源于它的10像素宽的矩形，因此该“时间基准”（图像顺序数）为10倍以上。 </li><li>复制透射DAT的名单A（每部电影一个列表），以Excel文件。平均值的指示剂染料刷新的几部电影中获得的透光率时程。产生由0.1乘以图像序列号一个实时的基础。保存的平均时间过程（两列）到一个文本文件。注：平均前，如果需要的话，画出各个时间的课程，拒绝任何违规行为。确保电影包含的指示剂染料的稳态透光率至少为5最终秒。 </li></ol></li><li>启动Matlab的拟合程序P F拟合（即“指标拟合”面板， 图3）来计算的摩尔渗透压浓度的时间过程中的各种参数。注意：根据在浴溶液的已知的初始和最终浓度，溶液的改变渗透压浓度的时间过程是从浓度时程来计算（计算值，反过来，从指示剂染料透射率），假设它遵循相同的动力学作为染料浓度。 P F飞度可以免费使用的程序。在“PfFit_Installer_web.exe”可以下载来自：P F飞度用户提供详细的解释和定义指南“是通过天哪访问的补充文件，这有助于熟悉的P F飞度程序的用户。 </li><li>在“指标飞度”面板，导入指示剂染料透过率（“指标数据文件&#39;， 图3A）的平均时间过程的数据和手动插入目前的实验参数和参数”宽度“和”初始猜测t_half“描述的指示剂染料浓度（ 图3B的时间过程。单击”运行“，以查看指示剂染料浓度随时间的变化的曲线（真实数据和合适的， 图4A）和模拟（计算）浴渗透压（ 图4B）注：股份公司洪水适合的数据是必要的（建议：开始在图3所示的值）。 </li></ol> 6，确定利用Matlab拟合程序P F飞度的P F 注：除基本假设对于原生质体作为一个真正的，完善的渗压计11的行为，P F的决心靠在假定P F可能会随时间而改变，这对P F这个动态underlies时间细胞体积变化的过程和该三个参数足以描述它：对网络连接 （的P F的初始值），边坡PF（的P F的线性变化率）和延迟（从浴中的起始处的周期摩尔渗透压浓度变化至电池体积变化的开始）。不同的模型可以测试，包括这些参数的不同组合和他们的价值观，包括空值11。 P <suB&gt;˚F适合搜索这些参数的最佳组合，得到-通过计算-最忠实地再现了细胞体积变化11中，计算出的试验时间，当然，反过来，从导入的一系列的细胞轮廓区域（见还补充&#39;P F适合使用说明书“）。 <ol><li>切换到“卷飞度”面板（ 图5）。选择要导入的区域的数据文件（与所分析的原生质体， 图5A中的“区域”的时间过程的文本文件）。选择“最后指示器底座”作为参数源（ 图5B;请参阅替代品“P F适合使用说明书”）。请注意：这些参数（ 图5D），然后用来再生在浴中用于装配过程的卷的渗压变化。 </li><li>在“音量飞度”面板（ 图5C），初始化（填写初始猜测的）的P F参数：P F，斜率PF和延迟（建议：从1开始，1和30，分别），之所以选择模型“类”（建议：首先II和标志“检查”的所有三个参数来安装）。点击“运行”，然后眼球的临时数字（ 图5E），并根据需要进行调整的延迟参数和记录的长度，。 </li><li>检查的结果曲线图（ 图6），以评估拟合的质量，并记录适当的错误。修改初始化参数了几折的每个，并重新&#39;Run“。注意：不要在程序卡住不要气馁 - 只要重新启动程序！ </li><li>重复此过程数次，开头的初始化参数的不同组合，其目的为拟合误差的最低值。 </li><li>直接从屏幕上复制的拟合结果列表，或者找出它们在T他PfFit产生的“_FIT_Vol_Results.txt&#39;文件。 </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+localisation+and+electrophysiological+characterisation+of+ion+channels+heterologously+expressed+in+a+plant+context.">A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context.</a> Plant Methods. 19, (2005).</li><li>Shoseyov, O., Posen, Y., Grynspan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Recombinant+Type+I+Collagen+Produced+in+Plants.">Human Recombinant Type I Collagen Produced in Plants.</a> Tissue Eng Part A. 19, 1527-1533 (2013).</li></ol>

We have nothing to disclose.

这里描述的是测量分离的植物原生质体的P F，适用的原则也适用于其他的球形细胞， 如 ，蛙卵母细胞11一个简单而非常有效的方法。这个方法是基于测量的P F响应于渗透压挑战到细胞。相反，基于这种方法的其它方法，然而，溶液的渗透压摩尔浓度的变化， 即 ，不是瞬时的，而是渐进的，在一个恒定的浴灌注，首先是等渗的溶液，其中，所述基线细胞体积?...

跨细胞膜的吸水率以及流量为植物存在于两者的细胞和整个植株水平的基本要求。在细胞水平上，水通道蛋白（水通道蛋白）发挥细胞膜1-3的渗透水渗透系数（P F）的调节中发挥关键作用。 至今，几种方法已被用于测定原生质体的内源P F从不同植物器官（ 如根，叶肉，内皮等 ，由肖蒙等 4中综述）。一种测量P F的方法之一是将原生质体暴露于渗透挑战，并监测其体积变化的初始速率（ 即 ，体积变化的早期线性相的斜率）。两种不同的方法根据该方法，以前描述的，两者都基于解决方案的瞬时交流。第一种是由immobiliz的荷兰国际集团的吸入微量的原生质体和开关的溶液流5和转移的原生质体从一种溶液到另一个使用微量6中的第二个。这些微量吸管和微量转移的方法，它允许在快速的解决方案交流的一开始图像采集（采集量变化的早期线性相位），可能涉及到的物理应力原生质体，并且需要专门的设备和专家的显微操作。 这里所描述的方法最大限度地减少了干扰的细胞，不涉及显微和允许的P F推导当浴灌注不是瞬时的。 酶消化后，将原生质体，浸没在等渗溶液中，被固定在有机玻璃的盖玻片玻璃底部（又名有机玻璃或有机玻璃）腔室通过电荷相互作用。然后，在一个恒定浴灌注，等渗溶液用低渗溶液产生低渗挑战原生质体冲走。原生质体的膨胀是视频记录，然后，通过结合约浴灌注的时间过程和细胞肿胀的时间过程中的信息时，P f由图像处理和曲线拟合程序确定。 这种方法的优点是，该实验是非常有效的， 即有可能在一个单一的检测同时监视几个细胞，并且它不需要特殊的设备或特别的显微操作技能。几个应用本方法也是可能的。例如，测定各种不同组织和植物，如叶肉细胞的天然P F和束从拟南芥叶片7，玉米叶肉或根皮层细胞8-10或悬浮培养的细胞11,12鞘细胞。在additi上，所以能够确定球形动物细胞的P F如卵母细胞11。另一个例子涉及到检查AQP活性通过基因的原生质体（或任何其他基因，其可能影响他们; 例如 ，激酶的基因）的瞬时表达及其对P F的贡献和判定;例如，番茄水通道蛋白SlTIP2的表达; 2拟南芥原生质体用PEG转化和决心SlTIP2; 2相关P F 13。最后，检查在不同的分子/物质P F（药物，激素等）的影响加入到溶液也进行检查，例如在水通道蛋白阻滞剂升汞 7。 以下方案描述拟南芥叶肉细胞和测定它们的P F的原生质体的分离。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:288px;">KCl</td>
			<td style="width:140px;">Chem-Impex International</td>
			<td style="width:159px;">01247-1</td>
			<td style="width:516px;"><a href="http://www.chemimpex.com/">http://www.chemimpex.com</a> 
			Any source, anal. grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:288px;">CaCl2</td>
			<td style="width:140px;">Merck</td>
			<td style="width:159px;">11718006</td>
			<td><a href="http://www.merck.com/">http://www.merck.com</a> 
			Any source, anal. grade</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:288px;">2-(N-morpholine)-ethanesulphonic acid (MES)</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">15152002</td>
			<td>http://www.sigmaaldrich.com 
			Any source, anal. grade</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:288px;">D-Sorbitol</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">18032003</td>
			<td>http://www.sigmaaldrich.com 
			Any source, anal. grade</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:288px;">Cellulase</td>
			<td style="width:140px;">Worthington, Lakewood, NJ, USA</td>
			<td style="width:159px;">LS002603</td>
			<td><a href="http://www.worthingtonbiochem.com/">http://www.worthingtonbiochem.com</a></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:288px;">Pectolyase</td>
			<td style="width:140px;">Karlan,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Phonix, AZ, USA</td>
			<td style="width:159px;">8006</td>
			<td><a href="http://www.karlan.com/">http://www.karlan.com</a></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:288px;">Polyvinyl-pyrrolidone K 30 (PVP)</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">81420</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:288px;"><a href="https://my.labguru.com/catalog/materials/216">Bovine Serum Albumin (BSA)</a></td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">A9418-5G</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:288px;">Protamine sulphate</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">P4380</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Poly-L-Lysine</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">P8920</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Xylene cyanol</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">X4126</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Silicone vacuum grease heavy</td>
			<td style="width:140px;">Merck</td>
			<td style="width:159px;">107921</td>
			<td><a href="https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&amp;SEO_ErrorPageOccurred=true&amp;attachments=CoA">https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&#38;SEO_ErrorPageOccurred=true&#38;attachments=CoA</a></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Inverted microscope&#160;</td>
			<td>Nikon</td>
			<td>Eclipse TS100/TS100F</td>
			<td><a href="http://www.nikoninstruments.com/">http://www.nikoninstruments.com</a></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Peristaltic pump</td>
			<td>BIO-RAD</td>
			<td style="width:159px;">EP-1 Econo&#160;Pump</td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Grayscale digital camera</td>
			<td>Scion Corporation</td>
			<td style="width:159px;">CFW-1308M</td>
			<td><a href="http://www.scioncorp.com/">http://www.scioncorp.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CMU 1394 Camera Driver&#8217; plugin for ImageJ</td>
			<td style="width:140px;">Carnegie Mellon</td>
			<td></td>
			<td><a href="http://www.cs.cmu.edu/~iwan/1394/download.html">http://www.cs.cmu.edu/~iwan/1394/download.html</a> 
			Free software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:140px;">NIH</td>
			<td></td>
			<td><a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> 
			Free software</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Econo Gradient Pump Fittings Kit</td>
			<td style="width:140px;">BIO-RAD</td>
			<td style="width:159px;">731-9006</td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Connectors, manifold</td>
			<td style="width:140px;">DirectMed</td>
			<td></td>
			<td><a href="http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S">http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Burette infusion sets (columns)</td>
			<td style="width:140px;">Welford</td>
			<td style="width:159px;">IF-BR-001</td>
			<td>http://www.welfordmedical.com/content.php?id=61</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tubing</td>
			<td style="width:140px;">TYGON</td>
			<td style="width:159px;">R-3603</td>
			<td><a href="http://www.usplastic.com/">http://www.usplastic.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Plexiglass slide etc.</td>
			<td style="width:140px;">Perspectiv</td>
			<td></td>
			<td><a href="http://www.perspectiv.co.il/index-en.html">http://www.perspectiv.co.il/index-en.html</a> 
			Our slide was custom-made, it does not appear on the web site but a copy can be remade to order as &#39;a copy of the slide already made for M. Moshelion&#39;.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3M packaging Scotch tape 1&#39;&#39;, clear</td>
			<td style="width:140px;">Viking Industrial, UK</td>
			<td style="width:159px;">VKMONO25</td>
			<td><a href="http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8">http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8</a> 
			any clear adhesive tape (sellotape, etc.) is likely to be OK</td>
		</tr>
	</tbody>
</table>

measuring the osmotic water permeability coefficient (pf) of spherical cells: isolated plant protoplasts as an example

研究水通道蛋白调控机制是水的关系的同时在细胞和整个植株水平的理解是至关重要的。这里介绍的是用于渗透水渗透系数（P f）如植物原生质体，适用于原则也适用于其他的球形细胞，如蛙卵母细胞的判定的简单和非常有效的方法。在测定法的第一步骤是从通过酶消化感兴趣的植物组织原生质体的分离与适当的等渗溶液的腔室。第二步骤包括渗透压挑战测定：原生质体固定在所述腔室的底部是通过低渗溶液提交的恒定灌注起始用的等渗溶液和遵守。细胞肿胀是录制的视频。在第三步骤中，图像脱机进行处理，以产生体积变化，体积变化的时间过程密切相关，在osmola的变化的时间过程RITY（安全）腔灌注介质，使用MATLAB写的（以下简称“PfFit&#39;）的曲线拟合程序，以产生P F。

为了确定在P f和比较不同的通道蛋白的活性，从 ​​拟南芥叶子叶肉原生质体中使用。这些原生质体中发现有低基础（背景）P F等级7，可以作为功能性表达系统，以使重现的P F测量。 从6周龄拟南芥植物成熟叶片原生质体分离和三个基因结构与水通道蛋白基因的拟南芥（AtPIP2 1）和玉米（ZmPIP1; 2 ZmPIP2; 4）瞬时（并单独）使...

Watch this Scientific Journal Video about Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example at JoVE.com

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

测量细胞的渗透水的渗透系数（P F）可以帮助理解水通道蛋白的调节机制（水通道蛋白）。在这里介绍的球形植物细胞原生质体P F决心涉及原生质体的分离和体积变化的过程中不断的浴灌注渗透挑战，因此他们的初始速度的数值分析。

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

measuring-osmotic-water-permeability-coefficient-pf-spherical-cells

Research

JoVE Journal

Biology

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

15.9K 次观看.  The Hebrew University of Jerusalem. 测量细胞的渗透水的渗透系数（P F）可以帮助理解水通道蛋白的调节机制（水通道蛋白）。在这里介绍的球形植物细胞原生质体P F决心涉及原生质体的分离和体积变化的过程中不断的浴灌注渗透挑战，因此他们的初始速度的数值分析。 

视频: Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

渗透避免IN线虫：突触功能的两个基因，人类同源 NRXN1 NLGN1，作为候选人自闭症

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

科研

生物学

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

表征草食动物的耐药机制：Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

渗透离心技术质外体中提取植物液使用叶<em&gt;菜豆</em&gt;为例

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

分析蛋白导入进强调，从植物中分离叶绿体

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...