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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<li>Shoseyov, O., Posen, Y., Grynspan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Recombinant+Type+I+Collagen+Produced+in+Plants%5BTitle%5D)+AND+%22Tissue+Eng+Part+A%22%5BJournal%5D)">Human Recombinant Type I Collagen Produced in Plants.</a> Tissue Eng Part A. 19, 1527-1533 (2013).</li>
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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><tbody><tr><td>KCl</td><td>Chem-Impex International</td><td>01247-1</td><td>&lt;a href=&quot;http://www.chemimpex.com/&quot;&gt;http://www.chemimpex.com&lt;/a&gt;&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>11718006</td><td>&lt;a href=&quot;http://www.merck.com/&quot;&gt;http://www.merck.com&lt;/a&gt;&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>2-(N-morpholine)-ethanesulphonic acid (MES)</td><td>Sigma</td><td>15152002</td><td>http://www.sigmaaldrich.com&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>D-Sorbitol</td><td>Sigma</td><td>18032003</td><td>http://www.sigmaaldrich.com&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>Cellulase</td><td>Worthington, Lakewood, NJ, USA</td><td>LS002603</td><td>&lt;a href=&quot;http://www.worthingtonbiochem.com/&quot;&gt;http://www.worthingtonbiochem.com&lt;/a&gt;</td></tr><tr><td>Pectolyase</td><td>Karlan,&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Phonix, AZ, USA</td><td>8006</td><td>&lt;a href=&quot;http://www.karlan.com/&quot;&gt;http://www.karlan.com&lt;/a&gt;</td></tr><tr><td>Polyvinyl-pyrrolidone K 30 (PVP)</td><td>Sigma</td><td>81420</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>&lt;a href=&quot;https://my.labguru.com/catalog/materials/216&quot;&gt;Bovine Serum Albumin (BSA)&lt;/a&gt;</td><td>Sigma</td><td>A9418-5G</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>Protamine sulphate</td><td>Sigma</td><td>P4380</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P8920</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>Xylene cyanol</td><td>Sigma</td><td>X4126</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>Silicone vacuum grease heavy</td><td>Merck</td><td>107921</td><td>&lt;a href=&quot;https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&amp;amp;SEO_ErrorPageOccurred=true&amp;amp;attachments=CoA&quot;&gt;https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&amp;amp;SEO_ErrorPageOccurred=true&amp;amp;attachments=CoA&lt;/a&gt;</td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Nikon</td><td>Eclipse TS100/TS100F</td><td>&lt;a href=&quot;http://www.nikoninstruments.com/&quot;&gt;http://www.nikoninstruments.com&lt;/a&gt;</td></tr><tr><td>Peristaltic pump</td><td>BIO-RAD</td><td>EP-1 Econo&amp;nbsp;Pump</td><td>&lt;a href=&quot;http://www.bio-rad.com/&quot;&gt;http://www.bio-rad.com&lt;/a&gt;</td></tr><tr><td>Grayscale digital camera</td><td>Scion Corporation</td><td>CFW-1308M</td><td>&lt;a href=&quot;http://www.scioncorp.com/&quot;&gt;http://www.scioncorp.com&lt;/a&gt;</td></tr><tr><td>CMU 1394 Camera Driver&amp;rsquo; plugin for ImageJ</td><td>Carnegie Mellon</td><td></td><td>&lt;a href=&quot;http://www.cs.cmu.edu/~iwan/1394/download.html&quot;&gt;http://www.cs.cmu.edu/~iwan/1394/download.html&lt;/a&gt;&lt;br/&gt; Free software</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij/&quot;&gt;http://rsb.info.nih.gov/ij/&lt;/a&gt;&lt;br/&gt; Free software</td></tr><tr><td>Econo Gradient Pump Fittings Kit</td><td>BIO-RAD</td><td>731-9006</td><td>&lt;a href=&quot;http://www.bio-rad.com/&quot;&gt;http://www.bio-rad.com&lt;/a&gt;</td></tr><tr><td>Connectors, manifold</td><td>DirectMed</td><td></td><td>&lt;a href=&quot;http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S&quot;&gt;http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S&lt;/a&gt;</td></tr><tr><td>Burette infusion sets (columns)</td><td>Welford</td><td>IF-BR-001</td><td>http://www.welfordmedical.com/content.php?id=61</td></tr><tr><td>Tubing</td><td>TYGON</td><td>R-3603</td><td>&lt;a href=&quot;http://www.usplastic.com/&quot;&gt;http://www.usplastic.com&lt;/a&gt;</td></tr><tr><td>3M packaging Scotch tape 1&#039;&#039;, clear</td><td>Viking Industrial, UK</td><td>VKMONO25</td><td>&lt;a href=&quot;http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8&quot;&gt;http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8&lt;/a&gt;&lt;br/&gt; 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

16.0K 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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

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 ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

多井板膜插入系统中3D 组织模型渗透扩散的测定与模拟方法

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

生物工程

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 ...

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. ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

大豆原生质体分离的简易方法及其在瞬态基因表达分析中的应用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

植物细胞类型的分离和转录组分析

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

玉米叶肉原生质体的可扩展转染

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

<meta charset="utf8"></head><body>用于研究耐洪机制的高效卷心菜原生质体系统

Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia stress. To identify mechanisms of flooding tolerance in cabbages, a versatile platform for genetic functional studies is needed to overcome the transformation-recalcitrant nature of cabbages. In this study, a cabbage protoplast transient expression system and a corresponding protoplast hypoxia induction protocol were developed. This protocol achieved a high yield and integrity of protoplast isolation from cabbage leaves, with a transfection efficiency exceeding 40% using optimized enzymatic conditions. To alleviate potential hypoxic influence before treatments, the W5 solution was bubbled with oxygen gas to increase dissolved oxygen levels. Several chemicals for adjusting oxygen levels and physiological oxygen-scavenging treatments were tested, including EC-Oxyrase, OxyFluor, sodium sulfite, and an oxygen absorber pack. Dual-luciferase assays showed that promoters of anaerobic respiration response genes BoADH1 and BoSUS1L were activated in cabbage protoplasts after hypoxia treatments, with the highest induction level observed after treatment with the oxygen absorber pack. In summary, the cabbage protoplast transient expression system combined with hypoxia treatment demonstrates an efficient and convenient platform. This platform can facilitate studies of gene function and molecular mechanisms associated with hypoxia responses in cabbages.

<meta charset="utf8"></head><body>用于研究芸苔属植物耐缺氧性的卷心菜原生质体系统的开发

This protocol describes an efficient cabbage mesophyll protoplast system. Various oxygen-deficient treatments were tested, and the system showed a high activation of hypoxia-responsive genes, facilitating studies of the genetic and molecular mechanisms of flooding tolerance in Brassicaceae vegetables.

用于研究芸苔属植物耐缺氧性的卷心菜原生质体系统的开发

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia ...

测量渗透水渗透系数（P

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