感谢Christopher Antos博士对文本的有用评论。这项工作得到了上海科技大学授予温妮·舒姆的启动资金和中国国家自然科学基金资助（NNSFC第31471370号）的资助。

所有动物实验均按照符合当地和国际要求的上海科技大学机构动物护理与使用委员会的指导方针进行。 实验动物<ol><li>使用8-12周龄的成年雄性Sprague-Dawley大鼠（约300-450g）。在大鼠的这个年龄，精子已经到达了马尾附睾。 </li></ol> 2.大鼠Cauda上皮细胞的分离注意:除非另有说明，以下步骤在非无菌条件下进行。 <ol><li> 准备解剖仪器和试剂 <ol><li>通过浸泡在70％乙醇中并使其风干来消毒解剖工具。 </li><li>打开温水浴（32°C）;准备并预热500毫升1x Roswell Park纪念研究所1640培养基（RPMI），补充1％（v / v）抗生素（100 U / mL青霉素和100μg/ mL链霉素最终），标记为"RPMI（+ P / S 1:100）"。在清洁的气流控制工作站中执行此步骤。 </li><li>准备并预热1x 500mL含有非必需氨基酸（0.1mM）和丙酮酸钠（1mM）的Iscove&#39;s Modified Dulbecco&#39;s Medium（IMDM），并补充有5-α-二氢睾酮（1nM），10％胎牛血清，1％（v / v）抗生素（100 U / mL青霉素和100μg/ mL链霉素最终），并标记为"全IMDM"。创建50 mL等分试样并用石蜡膜密封;储存于4°C。使用无菌条件。 </li><li>通过将胶原酶I型和II型胶原酶溶解在RPMI（+ P / S 1:100）中制备胶原酶消化溶液，得到溶液中每个胶原酶1 mg / mL。过滤0.22μm膜，标记为"胶原酶溶液"。保持在室温（RT）直到使用。调整酶溶液的体积关于酶的重量;来自单一大鼠的马尾uda附睾剂所需的最小体积为2 mL。 </li><li>用RPMI（+ P / S 1:100）填充35毫米的盘子。 </li></ol></li><li> 解剖大鼠马尾附睾 <ol><li>通过使用戊巴比妥钠85mg / kg ip或使用异氟醚室来灭绝动物，直到动物对尾部刺激刺激不起反应;遵循颈椎脱位。 </li><li>用70％乙醇擦拭消毒下腹部，轻轻将两只睾丸推至下腹部，然后打开阴囊附近的下腹部。 </li><li>拿起附睾脂肪，解剖出整个生殖器官（睾丸，附睾和输精管），并用RPMI（+ P / S 1:100）浸泡在盘中。 </li><li>将RPMI（+ P / S 1:100）的生殖器官转移到无菌工作站。 </li><li>从结缔组织和脂肪组织和表皮解剖出马尾附睾干燥胶囊。将一个附睾与约0.2mL的胶原酶溶液置于1.5mL管中。在干净的气流控制工作站中进行此步骤。 </li></ol></li><li> 单细胞从大鼠马尾附睾的分离 <ol><li>使用精细剪刀切割胶原酶溶液中的附睾，直到组织变成糊状液体。用1.5 mL管中的其余（〜0.8 mL）酶溶液轻轻冲洗剪刀。 </li><li>将管放置在金属温热混合器上，在37℃下振荡速度为1000rpm。 </li><li>将酶组织混合物在室温下以30xg离心3分钟，并倾析出主要含有精子的粘性上清液。 </li><li>将沉淀重悬于1mL Full-IMDM中以淬灭所有的酶活性。将细胞悬浮液转移到含有49mL RPMI（+ P / S 1:100）的50mL管中。 注意:任选地，通过具有恒定曲线的100μm网眼膜过滤细胞悬浮液离子以避免大的细胞聚集。然而，如果需要细胞悬浮液用于生长细胞单层，则不要使用网状过滤器。 </li><li>将细胞混合物在室温下以30xg离心10分钟;倾倒上清液。 </li><li>将沉淀重悬于1mL Full-IMDM中，温和研磨至少5分钟，以从酶处理的附睾组织混合物中解离单细胞。 </li></ol></li><li> 在无菌条件下从其他细胞中分离上皮细胞 <ol><li>在含有Full-IMDM的10cm培养皿上培养细胞悬浮液至少8小时或过夜，在32℃，5％CO 2的培养箱中培养。 </li><li>通过浸入100％酒精中预先准备无菌盖玻片。空气干燥并浸入少量的培养基中。将盖玻片放在6厘米培养皿或24孔板的单孔中。 </li><li>第二天早晨，通过轻轻收集细胞悬液来收获解离的上皮细胞培养皿主要由上皮细胞组成。将细胞悬浮液在室温下以30xg离心5分钟，然后倒出上清液。 </li><li>将细胞沉淀重悬于〜2mL全IMDM。 </li><li>将0.2mL收获的细胞悬浮液种植到每个无菌盖玻片的中心。 </li><li>让细胞悬浮液沉淀在液滴中至少10分钟，以使细胞松散地粘附到玻璃盖玻片上。小心地在10厘米盘的边缘加入1毫升的全IMDM，或在24孔板的每个孔中加入0.3毫升的全IMDM;不要打扰细胞。 </li><li>将分离的单个细胞在培养箱中的盖玻片上，在32℃，5％CO 2中直到膜片钳实验。 </li></ol></li></ol> 3.记录解决方案和微量移液器注意:对于膜片钳实验，请使用最优质的化学品和解决方案。 <ol><li> 准备库存解决方案 <ol><li>高压灭菌所有的瓶子库存储存和过滤所有的库存溶液（腐蚀性溶液除外），并在使用前过滤0.22μm膜。 </li><li>在室温下预先准备所有储备溶液，并储存在4 o C:5 M NaCl; 1 M KCl; 100mM MgCl 2 ; 100mM CaCl 2 ; 200mM NaH 2 PO 4 ; 100mM EGTA（pH7.0，用KOH）。处理5 M NaOH，1M HCl和1M KOH储备液作为腐蚀性溶液。 </li></ol></li><li> 制备标准外用生理盐水溶液（PSS） <ol><li>在膜片钳记录的早晨，将库存溶液温热至RT。 </li><li>除CaCl 2外 ，根据所需的最终体积吸取各种成分， 例如制备500 mL PSS:140 mM NaCl = 14 mL 5M; 5mM KCl = 2.5mL的1M; 1.2mM MgCl 2 = 6mL 100mM; 1.2mM NaH 2 PO 4 = 3mL 200mM。 </li><li>添加双倍去离子水（ddH 2 O）至最终体积为400mL并平衡。 </li><li>称量0.9 g葡萄糖和1.19 g HEPES，并完全溶解在溶液中。 </li><li>在搅拌下加入CaCl 2原料（2.5 mM = 12.5 mL，100 mM）。 </li><li>最多可达99％。 </li><li>使用NaOH或HCl将pH调节至7.4。 </li><li>检查渗透压，并根据需要使用5 M NaCl或葡萄糖进行调整。 </li><li>在圆筒中加入ddH 2 O至最终体积为500 mL。 </li></ol></li><li> 微量移液器内部溶液的制备（低EGTA K +溶液） <ol><li>根据所需的最终体积和浓度称量或移取每种试剂的正确体积， 例如用于制备50 mL低EGTA K +的细胞内溶液至体积约30 mL ddH 2 O:100 mM K-葡萄糖酸盐= 1.17g; 35mM KCl = 1.75mL 1M; 2mM MgCl 2 = 1mL100mM; 0.1mM EGTA = 0.05mL的100mM; 10mM HEPES = 0.072g。 </li><li>加入最终体积的约95％的足够水，并使溶液在室温下平衡。确保解决方案清晰。 </li><li>在不断搅拌溶液的同时，用KOH调节pH至7.2。 </li><li>称量并向溶液中加入0.078 g Mg-ATP，直至溶液完全溶解。 </li><li>将溶液置于冰上，并使用少量等分试样测量渗透压;通常，解决方案可测量〜290 mOsmol，不需要调整。如果渗透压与280-295 mOsmol显着不同，请制备新的溶液。 </li><li>将ddH 2 O添加到最终体积。 </li><li>将溶液分成500μL等分试样，用0.2μm注射器过滤器过滤，密封并立即保存在-20°C。 </li><li>在膜片钳实验之日，将一半的细胞内溶液在冰上解冻，并在膜片钳实验期间保持冷冻防止退化。 </li></ol></li><li>从玻璃毛细管拉出补片移液器（使用移液器拉拔器用户手册），以获得当充满细胞内溶液时具有5-10M电阻的微量移液器尺寸。 </li></ol> 4.设置补丁夹实验，建立细胞全细胞配置<ol><li> 设置膜片钳实验 <ol><li>打开膜片钳设置（电脑，电脑控制放大器，数字化仪等 ） </li><li>打开补丁钳软件（ 例如 AXON pCLAMP10或HEKA PatchMaster），并设置电生理记录协议。将信号滤波器设置为1-3 kHz的低通，数字转换器设置为10-20 kHz。 </li><li>打开相机，显微操纵器和光源。 </li><li>当计算机控制放大器打开时，通过用手接触实验者的身体接地的贴片钳，在触摸前台之前，为了保护它免受电击。 </li><li>将玻璃盖玻片上的培养上皮细胞转移到装有〜1mL标准PSS的记录室中。在进行膜片钳实验之前，使用移液器至少两次更换沐浴PSS。 注意:可选，根据计划的实验，使用标准PSS或其他外部溶液填充灌注系统。在开始进行膜片钳实验之前，以〜2 mL / min的速度将显微镜安装的记录室（RC-26G或RC-26GLP）与PSS进行几次。确保灌注系统中没有气泡被捕获。 </li><li>使用配有差分干涉对比度光学系统的10X和40X物镜在倒置显微镜下查看并选择细胞。寻找大型单独的细胞进行记录。通过粗糙m的球形识别孤立的附睾上皮细胞微绒毛在膜的一端和细胞内含量的极化分布（ 图1 ）。 </li><li>使用1 mL注射器（自制的非金属微量注射针），用内部溶液（低EGTA K +溶液，参见步骤3.3）填充微量移液管。确保微量移液管中没有气泡，这可能会增加微量移液器的阻力。使用足够的溶液，使内部溶液将氯化物涂覆的银线电极浸入微量吸管支架内。 </li><li>将微量移液管安装在电极夹中，并施加低正压（〜0.2 mL注射器体积）。保持低正压连续，直到在后续步骤中接触细胞膜。 </li></ol></li><li> 用记录单元建立全单元配置 <ol><li>以最高速度的微操纵器将移液器浸入浴液中。找到移液器提示在屏幕上连接数码相机;将显微操纵器速度降低到中高档模式。 </li><li>使用数据采集接口命令（ 例如 AXON系统中的"膜测试"），通过施加从计算机控制的放大器产生的电压步长（ 例如， 5毫秒为100毫秒），快速检查微量移液器电阻（5-10MΩ）。如果电阻显着超出此范围，请更换为新的微量移液器。 </li><li>开始向下移动显微镜上安装的物镜;逐渐将微量移液管引向选定的细胞。首先要降低目标，然后将微量移液器降低到焦平面，直到微量移液管位于所选细胞的中心表面之上。 </li><li>使用软件的命令器界面中的"pipette offset"命令，将移液器和液体溶液之间的液体接点取消为零。 </li><li>设置计算机控制放大器通讯安装到电压钳和膜测试到"Bath"模式。 </li><li>精细对焦以更清晰地观察细胞，然后在低中速度下使用显微操纵器逐渐降低微量移液管。 </li><li>当微量移液管接近细胞时（通过膜测试指令触发时由电流降低证明），立即取出低正压，施加弱负压（0.1 mL注射器体积）以形成igaseal（&gt; 1GΩ） 。 </li><li>用膜测试监测电阻。如果电阻&gt; 500MΩ但&lt;1GΩ，则施加负电位（通常作为保持电位设置为-60 mV），这有助于形成gigaseal。补偿微量移液器的瞬态电容电流。 </li><li>如果密封件&gt; 1GΩ并且稳定（如软件界面所示），请施加简短强力的吸力以破坏细胞膜。不要赔偿本金电阻和电池电容。 </li><li>在达到成功的全细胞配置之后，立即从-60mV的保持电位施加10mV超极化步骤（具有最小时间间隔，20ms持续时间，20kHz的信号样本的5个走线）。 </li><li>将电压模式切换到零电流模式，并标记软件界面的读数，或对电池的膜电位读数执行无间隙记录（10-60秒）。 </li><li>快速切换回并保持在电压模式，并根据计划的实验应用电压协议，并测量当前响应。在记录期间，减法不适用于本征泄漏电流。 </li><li>监视记录期间响应的稳定性或单元格的不同参数。例如，使用"膜测试"命令界面来检查输入电阻（R i ），串联电阻（R s ）和电池薄膜电容（C m ）在薄膜测试期间在"Cell"模式下的协议切换。 注意:输入阻力的突然下降通常表示松散的贴片，并且串联电阻的显着增加可能指示微量移液管尖端被细胞内细胞器或膜片段堵塞。在录制过程中，定期监测微量移液器位置，以检查是否发生漂移，这可能会导致补片丢失。如果漂移是一个问题，请小心地将微量移液管和修补池一起提起，远离记录室底部。这个步骤有时可能会导致贴剂的损失，在这种情况下，有必要重复全细胞膜片钳制程序。 </li></ol></li></ol> 5.细胞被动电生理特性分析<ol><li>在从膜片钳实验中获得数据后，在Clampfit软件中打开无间隙数据并测量平均值每个细胞的静息膜电位的值。或者，使用在当前模式下从零电流电压标记的值。校正具有液体结电位的值（本研究中为12.4 mV）。 </li><li>打开来自从细胞得到的10毫伏超极化步骤（ΔV）的数据，之前和期间的步骤测量电流差（Δ 我 步骤 ），并计算使用下式作为输入电阻（R I）: <img alt="公式1" src="/files/ftp_upload/55700/55700eq1.jpg" /></li><li>计算电池电容（C m ，以pF为单位）（使用与10 mV超极化步骤相同的电流数据，通过在电压阶跃触发时初始瞬态衰减电流下积分膜电容电流下的总面积）以获得单元的总累积电荷（Q，单位为pA·ms）的值。使用以下公式: <img alt="公式2" src ="/ files / ftp_upload / 55700 / 55700eq2.jpg"/&gt; </li><li>通过使用标准指数算法拟合10 mV负步骤的初始瞬态电流，计算每个单元的串联电阻（R s ）的值，以获得时间常数衰减电流（ T ，单位为ms），并使用以下等式: <img alt="公式3" src="/files/ftp_upload/55700/55700eq3.jpg" /></li></ol>

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作者没有什么可以披露的。

在该方案中，大鼠卡达附睾的酶分散始终产生健康的上皮细胞。用于膜片钳实验的附睾上皮细胞的质量取决于方案中的几个关键步骤。例如，在低离心力（30×g）下离心细胞混合物对于去除精子和附睾腔内含量是重要的;附睾上皮细胞在细胞培养中精子存在时变得不健康。另外，在培养皿上培养解离的细胞混合物数小时是去除成纤维细胞和平滑肌细胞的重要步骤;非上皮细胞在培养皿上粘附更快，因此，将上皮细胞留在悬浮液中，其可以通过温和抽吸再次收获。此外，用精细玻璃移液管消化和研磨胶原酶是释放的两个重要步骤单细胞在细胞解离过程中。最后，附睾细胞的酶消化对于膜片钳实验是至关重要的，因为通过低效的酶活性的消化不足或通过延长的孵育过度消化可能破坏移液管和细胞膜之间的千兆欧密封形成。 在细胞混合物中，具有较大的柱状形状的细胞在一端具有轴承立体结合并且细胞内内容物的极化分布可能是主细胞或透明细胞。不具有显着的微绒毛和极化细胞内容物的其它细胞可以包括基底细胞和来自免疫系统的一些细胞。除了描述附睾上皮细胞的基本形态特征外，该方案采用全细胞膜片钳方法测量每个细胞的被动膜性质。这些膜性质允许初步区分细胞类型。此方法的过程中连续记录19具有一定的局限性，如与移液管溶液与电子的变化相对于所述蜂窝结构修饰胞内内容冲洗，20。我们仅提供在建立全细胞配置后立即测量的参数，这反映了细胞的初始，相对完整的条件。每个细胞的被动膜性质包括膜电容，输入电阻和静息膜电位。 细胞膜的比电容通常认为为1μF/ cm 2并且这适用于细胞（如，免疫细胞和神经元）与它们的细胞膜16,18限定的折叠如此。然而，对于电池w，比电容常数趋于更大更复杂的细胞结构，如上皮细胞，其在不同的隔室中具有许多细胞膜折叠。报道指出，MDCK上皮细胞系值分别为4μF/ cm 2和3μF/ cm 2 ，分别为顶端和基底膜特异性电容17 。在本研究中，对于无微绒毛细胞组和主要细胞和透明细胞组（ 即微绒毛细胞），测量的平均膜电容分别为〜8pF和〜12pF。使用1μF/ cm 2的我们的计算的比电容，估计细胞表面积为500μm2和1,000微米2为无微绒毛细胞和微绒毛细胞，分别，其对应于为13μm和18的直径微米。然而，这些估计大于基于显微镜下的细胞尺寸的预期值，这是〜8对于无微绒毛细胞和微绒毛细胞，分别为1; m和〜12μm，这些细胞分别为约2.4μF/ cm 2和1.9μF/ cm 2 。我们的研究结果表明，非微绒毛细胞的膜景观比微绒毛细胞更复杂，与其分泌和运输功能一致。 细胞膜的比电容通常认为为1μF/ cm 2并且这适用于细胞（如，免疫细胞和神经元）与它们的细胞膜16,18限定的折叠如此。然而，对于具有更复杂的细胞结构（如上皮细胞）的细胞，比电容常数倾向于更大，其在不同隔室中具有许多细胞膜折叠。报告显示MDCK上皮细胞系值为4μF/ cm 2和3μF/ cm 2 </sup&gt;针对顶端和基底膜特异性电容分别为17 。在本研究中，对于无微绒毛细胞组和主要细胞和透明细胞组（ 即微绒毛细胞），测量的平均膜电容分别为〜8pF和〜12pF。使用1μF/ cm 2的我们的计算的比电容，估计细胞表面积为500μm2和1,000微米2为无微绒毛细胞和微绒毛细胞，分别，其对应于为13μm和18的直径微米。然而，这些估计大于基于显微镜下的细胞大小的预期值，对于无微绒毛细胞和微绒毛细胞分别为〜8μm和〜12μm，并且这些细胞与特定电容值更好地相容2μF/ cm 2 。这表明附睾上皮细胞的膜景观是more比其他细胞类型复杂，这与其分泌和运输功能一致。 在全细胞记录中，穿过膜的泄漏电导和膜和移液管之间的密封都有助于测量的膜性质。密封件具有比膜显着更高的电阻，因此对被动膜性质的测量（例如输入电阻和零电流膜电位）的影响最小。与该概念一致，在千兆欧门槛值下的密封电阻相对于每个电池的输入电阻的相关分析导致约0.02的值，表明非常弱的影响。密封电阻或输入电阻与静息膜电位或-100 mV时测量的电流值的进一步相关分析也为所有测试细胞或仅主母细胞提供了较低的值。我们的结果表明附睾主细胞和透明样细胞的输入电阻显着低于无微绒毛细胞，从而为鉴别细胞类型提供了初步参数。这些结果表明，主要细胞中的低输入电阻是细胞的固有生理特性。单个上皮细胞还具有响应于所应用的方案和实验条件的不同的电流模式。我们观察到每个定义的细胞类型在相同的施加电压方案下显示出独特的电流模式。 图3所示的例子显示了在准生理条件下从主要细胞记录的典型电流响应，如我们之前报道的9 。每种特殊细胞类型的电生理特征容易分化（本文中未显示数据）。基于这些参数，如结果所述，不同的细胞类型可以粗略地分类为主细胞，透明样细胞和无微绒毛细胞。 输入电阻是响应于从-60mV的保持电位引起的施加电位步骤（本研究中的10mV超极化步骤）的膜电阻。这反映了应用潜力对开放渠道活动的程度。在这方面，低电阻意味着电池的高内在"泄漏"电导，而高电阻意味着封闭的通道。主细胞中的低输入电阻值对应于突出的膜电导，而在测试膜电位下具有小电导的透明样细胞意味着在记录条件下其适度的电导。无微绒毛细胞的显着高的输入电阻意味着它们在这种状态下没有开放的通道。在零cu对于三个细胞组，测量的分离的上皮细胞的平均静息膜电位在26-33mV的范围内。这些值与使用微电极方法21的报道的膜电位（-30mV）相当。然而，尽管没有自发的尖峰电位，测量的静息电位在各个细胞中（从+ 3mV到-63mV）变化很大。这种变化可能反映了不同细胞类型的不同离子通道活性的相互作用，如主要细胞中TRPV6和TMEM16A通道的偶联相互作用，正如我们最近报道的9 。值得注意的是，当细胞用ATP移液管透析时，我们观察到主细胞中没有自发电位;这与上皮细胞的分类一致是不可兴奋的4 。当细胞用pI透析时的进行性超极化没有ATP的pette溶液可能反映了ATP敏感钾电流的逐渐出现。据报道，该ATP敏感性钾离子通道在大鼠附睾的主细胞的高尔基体中表达，并且细胞内ATP的消耗导致至K ATP通道14，22的激活。这一观察结果表明，ATP在吸管溶液中的含量有利于附睾主细胞的稳定生理条件。 全细胞膜片钳技术是通常用于研究兴奋细胞如神经元的电生理学的已知方法。在本文中，我们表明它也是一个有用的工具，用于表征非兴奋性细胞，如上皮细胞的生物电性质。此外，该协议允许对原发性孤立性附睾的功能调查上皮细胞进一步阐明其在附睾中的生理作用。这项研究提供了从大鼠cauda附睾分离的上皮细胞的一些初步电性质，以帮助未来对这些细胞的生理行为的研究及其在附睾中的潜在的生物相关性。

男性生殖道的附睾是一层衬有一层马赛克上皮细胞的器官。如在其他上皮组织中，附睾上皮的各种细胞类型，包括主要细胞，透明细胞，基底细胞和来自免疫和淋巴系统的细胞，以协调的方式起作用，作为小管前线的屏障，并且作为支撑为精子成熟和生理学1，2，3的细胞。因此，这些上皮细胞在生殖健康中起重要作用。 上皮细胞通常被视为不可兴奋细胞不能产生响应全或无动作电位去极化到刺激，由于缺乏电压门控Na +或Ca 2+通道4,5。然而，上皮细胞表达uni一组离子通道和转运蛋白调节其特殊的生理作用，如分泌和营养物质运输6 。因此，不同的上皮细胞具有特征的电性质。例如，主细胞表达的CFTR用于流体和氯化运输和表达对钙的重吸收的TRPV6，而明确细胞表达质子泵V-ATP酶对于管腔酸化1，7，8，9。调节附睾上皮细胞的生理特征的一些转运蛋白和离子通道已被报道，但附睾上皮细胞的功能特性在很大程度上尚未了解10，11，12，13。 瓦油细胞膜片钳记录是用于检查可兴奋细胞和不可兴奋细胞的固有特性的已知技术，特别有助于研究异源细胞样品中主要解离的细胞的功能;电压钳用于测量无源膜特性和单电池14，15的离子电流。被动膜性质包括输入电阻和电容。前一个参数表示内在膜电导，而后者则表示细胞膜的表面积（磷脂双层，其中离子通道和转运蛋白位于其中，其作为分离细胞外和细胞内介质的薄绝缘体）。膜电容与细胞膜的表面积成正比。与输入电阻反映的膜电阻一起，膜时间常数w可以确定细胞膜电位对离子通道电流的流动有多快。在这点上，由电流响应特性从一系列施加于细胞电压的步骤相结合，生物物理动力学和细胞的性质被确定15，16，17，18。 在本文中，我们描述了使用全细胞膜片钳从大鼠cauda附睾分离上皮细胞的程序和用于测量解离的细胞混合物中不同细胞类型的膜性质的步骤。我们显示附睾主细胞表现出不同的膜电生理特性，并且可以容易地从其他细胞类型鉴定电导。

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	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Instrument of AXON system</td>
			<td style="width:129px;"></td>
			<td style="width:139px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:149px;">Computer controlled amplifier</td>
			<td style="width:129px;">Molecular Devices - Axon</td>
			<td style="width:139px;">Multiclamp 700B patch-clamp amplifier</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Digital Acquisition system</td>
			<td style="width:129px;">Molecular Devices - Axon</td>
			<td style="width:139px;">Digidata 1550 converter</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Microscope</td>
			<td style="width:129px;">Olympus</td>
			<td style="width:139px;">BX-61WI</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Micromanipulator</td>
			<td style="width:129px;">Sutter Instruments</td>
			<td style="width:139px;">MPC-325</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Recording chamber and in-line Heater</td>
			<td style="width:129px;">Warner Instruments</td>
			<td style="width:139px;">TC-324C</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Instrument of HEKA system</td>
			<td style="width:129px;"></td>
			<td style="width:139px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Patch Clamp amplifier</td>
			<td style="width:129px;">Harvard Bioscience - HEKA</td>
			<td style="width:139px;">EPC-10 USB double</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Microscope</td>
			<td style="width:129px;">Olympus</td>
			<td style="width:139px;">IX73</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Micromanipulator</td>
			<td style="width:129px;">Sutter Instruments</td>
			<td style="width:139px;">MPC-325</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Recording chamber and in-line Heater</td>
			<td style="width:129px;">Warner Instruments</td>
			<td style="width:139px;">TC-324C</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Other Instrument</td>
			<td style="width:129px;"></td>
			<td style="width:139px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Micropipette Puller</td>
			<td style="width:129px;">Sutter Instrument&#160;</td>
			<td style="width:139px;">P-1000</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:149px;">Recording Chamber</td>
			<td style="width:129px;">Warner Instruments</td>
			<td style="width:139px;">RC-26G or homemade chamber</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:149px;">Borosilicate capillary glass with filament</td>
			<td style="width:129px;">Sutter Instrument / Harvard Apparatus</td>
			<td style="width:139px;">BF150-86-10</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Vibration isolation table</td>
			<td style="width:129px;">TMC&#160;</td>
			<td style="width:139px;">63544</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Digital Camare</td>
			<td style="width:129px;">HAMAMASTU</td>
			<td style="width:139px;">ORCA-Flash4.0 V2 C11440-22CU</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Reagents for isolation</td>
			<td style="width:129px;"></td>
			<td style="width:139px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">RPMI 1640 medium</td>
			<td style="width:129px;">Gibco</td>
			<td style="width:139px;">22400089</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">Penicillin/Streptomycin</td>
			<td style="width:129px;">Gibca</td>
			<td style="width:139px;">15140112</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="43" style="height:43px;width:149px;">IMDM</td>
			<td style="width:129px;">ATCC&#160;</td>
			<td style="width:139px;">30-2005&#160;</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="43" style="height:43px;width:149px;">IMDM</td>
			<td height="22" style="height:22px;width:129px;">Gibco</td>
			<td style="width:139px;">C12440500BT</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Collagenase I</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:139px;">C0130</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Collagenase II</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:139px;">C6885</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:149px;">5-&#945;-dihydrotestosterone</td>
			<td style="width:129px;">Medchemexpress</td>
			<td style="width:139px;">HY-A0120</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Fetal bovine serum</td>
			<td style="width:129px;">capricorn</td>
			<td style="width:139px;">FBS-12A</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:149px;">Micropipette internal solutions (K+-based solution) (pH 7.2, 280-295 mOsm)</td>
			<td style="width:129px;"></td>
			<td style="width:139px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">KCl, 35mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900068</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:149px;">MgCl2 &#183; 6H2O, 2mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">M2393</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">EGTA, 0.1mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">E4378</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">HEPES, 10mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900477</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">K-gluconate, 100mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">P-1847</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Mg-ATP, 3mM</td>
			<td style="width:129px;">Sigma/Various</td>
			<td style="width:139px;">A9187</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:149px;">The standard external recording physiological salt solution (PSS) (pH 7.4, 300-310 mOsm)</td>
			<td style="width:129px;"></td>
			<td style="width:139px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">NaCl, 140mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900058</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">KCl, 4.7mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900068</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:149px;">CaCl2, 2.5mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900266</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:149px;">MgCl2 &#183; 6H2O, 1.2mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">M2393</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:149px;">NaH2PO4, 1.2mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900060</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">HEPES, 10mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900477</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">Glucose, 10mM</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900392</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">For pH adjustment</td>
			<td style="width:129px;"></td>
			<td style="width:139px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">NaOH</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">V900797</td>
			<td style="width:207px;">Purity &#62;=97%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:149px;">KOH</td>
			<td style="width:129px;">Sigma/various</td>
			<td style="width:139px;">60371</td>
			<td style="width:207px;">Purity &#62;=99.99%</td>
		</tr>
	</tbody>
</table>

whole-cell patch-clamp recordings of isolated primary epithelial cells from the epididymis

附睾是精子成熟和生殖健康的重要器官。附睾上皮由复杂连接的细胞类型组成，不仅在分子和形态特征上而且在生理特性上都是不同的。这些差异反映了其各种功能，它们共同为睾丸内附睾精子发育建立必要的微环境。了解附睾上皮细胞的生物物理特性对于在生理和病理生理条件下显示其在精子和生殖健康中的功能至关重要。虽然其功能特性尚未完全阐明，附睾上皮细胞可以使用膜片钳技术进行研究，膜技术是测量单细胞的细胞事件和膜性质的工具。在这里，我们描述了细胞分离和全细胞膜片钳记录方法确定主要解离的上皮细胞从大鼠cauda附睾的电性质。

对于上皮细胞从大鼠马尾附睾隔离所描述的酶消化过程是从我们以前的研究9,12一个经修改协议。该方法产生具有超过90％存活力和无表面起泡或肿胀细胞体积的单细胞混合物。异质细胞混合物主要由主要细胞，透明细胞和基底细胞组成，如前所述1 。在该方案中，可以通过在培养皿上培养主要解离细胞过夜以获得附睾上皮细胞的相对纯的样品，以允许将非上皮细胞（如成纤维细胞和平滑细胞）粘附在培养皿上，如在图1A中示出 （收获前）。然后可以在显微镜下通过其表型来初步区分细胞类型并根据其特定的被动膜特性进行分类。在解离的细胞混合物中，存在一组在其表面膜的一端具有微绒毛或粗糙膜的细胞和极化的细胞内容物;这些被称为主细胞或透明样细胞（ 图1B ，箭头）。这些极化上皮细胞通过其形态特征是不可区分的，但可以根据从全细胞膜片钳记录获得的被动膜特性和电导反应来鉴定。另一组细胞的尺寸较小，膜的一端没有毛细血管细胞，没有明显的极化细胞内容物;这些被称为无微绒毛细胞，并且主要由基底细胞组成（ 图1B ，星号）。 主要上皮主要细胞可以与其他细胞区分开使用全细胞膜片钳技术响应于施加的电压方案，具有不同的被动膜性质（ 图2 ）和电流模式（ 图3 ）。细胞的被动膜电生理特性可以帮助评估个体细胞和细胞类型组的初始健康状况。 图2A给出了每个电池组的膜电容（C m ），输入电阻（R m ）和膜电位（V m ））的各个点图的总结。不同细胞类型（〜0.4 ms）之间的膜电容的时间常数没有差异（在本手稿中未显示数据）。使用低EGTA K +溶液，主要细胞的平均测量膜电容为9.4 0.5 pF（n = 32），而透明样细胞为9.7 1.9 pF（n = 12）。膜电容无微绒毛细胞为5.2±0.8pF（n = 17），统计学上小于原细胞和透明细胞膜电容。 图2A （中图 ）主电池的输入电阻为1.1±0.2GΩ（n = 32），透明电池的输入电阻为2.2±0.8GΩ（n = 12），明显低于（P &lt;0.001）无微绒毛细胞（8.9±2.1GΩ; n = 17）。 如图2A （右图）所示，在建立全细胞构型后不久测量零电流膜电位（ 即静息膜电位V m ），并用于比较校正后的组与液体结电位（12.4毫伏）。将细胞常规浸泡在标准PSS中，并用含有ATP的0.1mM EGTA K +移液管溶液透析。那个主要细胞的膜电位为-26±2（n = 28），介于-51 mV和+1 mV之间，透明细胞的平均膜电位为-30±3 mV（n = 10），在-47 mV和-17 mV之间。非微绒毛细胞具有最高的平均膜电位，其值为-33±5mV（n = 17），-63mV和-13mV之间。 主要细胞的低输入电阻表明在这些细胞中存在固有的电导，但它可以指示移液管和贴片细胞膜之间的密封泄漏。为了解决这个问题，我们首先从记录中突然改变（表示密封泄漏）排除了这些电池的数据。然后我们分析了在千兆欧门槛密封电阻与输入电阻之间的相关性， 如图2B所示 。结果是非常低的相关值（R 2≈0.02）。这表明密封电阻对输入电阻的影响可以忽略不计，并且所报告的各种电池的被动电性能（如主电池的低输入电阻）是这些电池的固有特性。 图3A是在准生理条件下记录的主要细胞的典型电流响应，其中细胞在正常生理盐溶液（PSS）中洗澡，用低EGTA K基移液管溶液透析并从-60mV的保持电位刺激到一系列500ms的测试电压（范围从-120mV到+60mV， 如图3B的插入物所示 ）。 图3C中的两个描绘示出了在零电流钳位下存在的或者移液管中没有ATP。在ATP的存在下，静息电位相对稳定。而当没有ATP的移液管溶液进行细胞透析时，观察到进行性超极化。用移液管溶液开始细胞透析时测量的初始静息膜电位显示在具有或不具有ATP的细胞中没有显着差异（ 图3D ），表明测量值仍然是完整的并且受到移液管溶液的最小影响。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55700/55700fig1.jpg" /> 图1 :分离的大鼠cauda附睾上皮细胞的形态特征。 （ A，B ）在（ A ）之前和之后（ B ）重新收获ove后从大鼠cauda附睾分离的上皮细胞的实例在膜片钳实验之前的菜上的rnight文化。箭头表示微绒毛细胞，其主要由主细胞和透明样细胞组成。星号表示无微绒毛细胞。仅选择单个细胞用于膜片钳记录。刻度棒=10μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55700/55700fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55700/55700fig2.jpg" /> 图2 :单一大鼠cauda附睾上皮细胞的被动膜性质。 （ A ）单个大鼠cauda附睾上皮细胞的被动膜性质的点图。移液溶液是低EGTA K +和洗浴溶液是标准PSS。 （ B ）输入资料的相关分析具有电池的密封电阻。 NS :无明显差异，P &lt;0.05，** P &lt;0.01 *** P &lt;0.001显着性差异相对于使用Bonferroni post-hoc检验的单因素方差分析的适当对照。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55700/55700fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55700/55700fig3.jpg" /> 图3 :单个附睾原代细胞中典型的全细胞电流。 （ A ）从准生理条件下记录的单个附睾上皮细胞记录的典型全细胞电流使用泛素B插图所示的脉冲诱导方案。虚线表示零电流水平。 （ B ）当前响应的电流 - 电压关系在指定的时间点测量，如A.数据是来自至少三只动物的八个主要细胞的平均值±SEM。 （ C ）用ATP（+ ATP）或无ATP（-ATP）用移液管溶液透析的主要细胞的静息膜电位的代表性追踪。 （ D ）条形图显示+ ATP和-ATP的初始静息膜电位之间没有显着差异。值是在图C中指示的时间点的平均值±SEM。条中括号内的数字表示来自至少三只动物的测试细胞数。 NS :无显着性差异。 （ E ）所有细胞或主要细胞的密封电阻和输入电阻相对于在全细胞建立后不久的零电流钳位下测量的静息膜和在超极化测量的当前幅度的相关分析从保持p步进至-100 mVotential。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55700/55700fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis at JoVE.com

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

我们提出了一种协议，结合细胞分离和全细胞膜片钳记录来测量主要解离的上皮细胞从大鼠cauda附睾的电性质。该方案允许研究原发性附睾上皮细胞的功能特性，以进一步阐明附睾的生理作用。

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

whole-cell-patch-clamp-recordings-isolated-primary-epithelial-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.9K 次观看.  ShanghaiTech University. 我们提出了一种协议，结合细胞分离和全细胞膜片钳记录来测量主要解离的上皮细胞从大鼠cauda附睾的电性质。该方案允许研究原发性附睾上皮细胞的功能特性，以进一步阐明附睾的生理作用。 

视频: Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

在肉瘤细胞间质上皮转变的诱导

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...