在生物分子筛查设施 (BSF), 洛桑进行了时间推移显微镜实验。我们感谢马克 Delachaux (服务 Audiovisuel, 洛桑) 的录像和编辑的电影。

注: 本研究采用 E14 ES 细胞系。然而, 该协议直接适用于任何其他的小鼠 ES 细胞系, 表达一种与 SNAP 标签融合的感兴趣的蛋白质, 或者通过标记内源蛋白或使用过度表现。在结果部分所示的例子中, 使用了强力霉素-诱导的 snap 标记融合细胞系 (snap 标记融合到以下蛋白质: Nanog, Oct4, Srsf11, 或对荧光蛋白 mOrange2 和 sfGFP, 并置于控制下强力霉素诱导的启动子。有关用于生成强力霉素诱导的 SNAP 标记融合细胞系的质粒的进一步信息, 请参见11 。强力霉素诱导的系统可以特别有用, 因为它能够严格控制的时间和强度的表达的蛋白质的利益。建议采用 c-终端定位, 因为改变 n 端氨基酸序列更有可能改变目标蛋白的半衰期 (n 端规则12)。
1. e-钙黏附素涂层和细胞播种
<ol><li>生成一个 ES 单元格线, 表示与快照标记4融合的感兴趣的蛋白质。</li>
	<li>100毫米细胞培养皿4毫升0.1% 明胶 (稀释磷酸盐缓冲盐水 (PBS), 没有 Ca2 +/毫克2 +) 为 1 h. 除去明胶, 让干燥 1 h. 立即使用或将涂覆的盘子储存多达2月。</li>
	<li>培养 es 细胞培养基中的细胞系 (格拉斯哥最低必需培养基, 辅以 10% es 细胞合格胎牛血清, 2 毫米丙酮酸钠, 1% 非必需氨基酸, 1% 青霉素/链霉素, 2 毫米 l-谷氨酰胺, 100 µM 2-基, 白血病抑制因子 (LIF), 3 µM CHIR99021 和0.8 µM PD184352) 在明胶涂层的菜肴在37° c 和 5% CO2 2 天, 直到到达一个汇合 10-20 的宇达细胞每道菜。 注意: 在这里, LIF 是由 HEK 293T 细胞的瞬态转染产生的, 其次是上清收集和过滤。每批 LIF 测试的潜力, 以保持多, 但浓度没有确定。然而, 重组 LIF 也可以在商业上使用, 并且通常用于小鼠 ES 细胞培养的浓度是1000单位/毫升13。</li>
	<li>外套一个96井板适合成像与30µL 重组小鼠 e-钙黏蛋白 fc 嵌合体蛋白质 (e-cad-fc) 每井 (5 ng/µL, 稀释在 PBS 与 Ca2 +/毫克2 +。使用库存浓度 100 ng/µL, 储存在-80 ° c)。避免广泛的移, 因为电子 cad Fc 是非常脆弱的。孵育在37° c 为1.5 小时。 注: 视显微镜而定, 可使用其他格式。</li>
	<li>吸气电子 cad Fc, 用100µL 的 PBS 与 Ca2 +/毫克2 +, 并添加100µL 预热 ES 细胞培养基。</li>
	<li>用5毫升 PBS 清洗感兴趣的细胞而不需要 Ca2 +/毫克2 +。吸气并加入2毫升的胰蛋白酶-EDTA 0.25%。孵育4分钟, 在37° c。添加4毫升 es 细胞培养基, 重和旋转 1000 x g, 4 分钟, 在2毫升的新鲜 es 细胞培养基中吸取上清和重颗粒。 注: 不含 ca2 +/Mg2 +的 PBS 应用于在 trypsinization 之前清洗单元格, 以删除 ca2 +离子, 这是细胞黏附所必需的。相比之下, 对于 e cad fc 稀释和涂层 (步骤 1.4), 使用 PBS 与 ca2 +/Mg2 +, 因为 ES 细胞与 e-cad-fc 涂覆的盘子的黏附力是 ca2 +依赖14。</li>
	<li>在 ES 细胞培养基的1毫升中稀释悬浮细胞 1:10, 使用计数室 (载荷10µL 为 0.1 mm 深度的腔)。</li>
	<li>种子3万细胞/cm2在电子 cad-Fc 涂层成像板。用 ES 细胞培养基填充200µL。对于强力霉素诱导的细胞系, 诱导与适当剂量强力霉素 (优化剂量和时间诱导提前。在这里使用的细胞系治疗 500 ng/毫升强力霉素24小时前成像)。孵育在37° c 和 5% CO2 , 并进行脉冲标记和成像24小时后, 细胞播种。</li>
</ol>2. 标签的脉冲标记
注意: 对于蛋白质衰变实验来说, 使用适当的染料浓度是至关重要的。浓度应足够高, 以产生一个明亮的信号, 在开始时的时间推移, 因为荧光将减少随着时间的推移。然而, 使用太高的染料浓度可能会导致残留染料留在培养基或在细胞中, 即使在洗涤后。在电影的过程中, 游离染料可能会绑定到新产生的 SNAP 标记分子, 这将扭曲衰变曲线。所观察到的荧光信号将取决于染料的性质、所使用的细胞线以及相应蛋白质的表达水平。因此, 通过测试不同的稀释, 从制造商对活细胞成像的稀释建议开始, 对染料浓度进行优化是至关重要的。本研究采用了一种远红光荧光基板。测定了强力霉素诱导的过度表达细胞株的 12 nM 的最佳浓度。
<ol><li>在预热 ES 细胞培养基中, 将该染料稀释到适当浓度。使用吸管轻轻地从先前在96井板上播种的细胞中去除培养基, 并在每个井中添加50µL 的稀释的对齐染料。孵育30分钟, 在37° c。</li>
	<li>用吸管吸去稀释后的染料, 用200µL 预热 PBS (不含 Ca2 +/Mg2 +) 洗涤3x。添加200µL ES 细胞培养基, 孵育15分钟, 在37° c。</li>
	<li>重复前面的洗涤步骤两次以上。 注: 为了去除任何未粘合的染料, 广泛的洗涤是重要的。重复洗涤步骤与 PBS 去除残留的胞外染料在培养基中, 而15分钟孵化确保在开始成像实验之前, 对捕捉标签融合蛋白的鲁棒标记。然而, 通过温和的移来执行洗涤步骤, 不要使用自动器, 因为在电子 cad Fc 上播种的细胞倾向于容易分离。</li>
	<li>添加200µL 成像介质。为减少荧光背景, 使用酚红游离培养基 (辅以 10% ES 细胞合格胎牛血清, 2 毫米丙酮酸钠, 1% 非必需氨基酸, 1% 青霉素/链霉素, 2 毫米 l-谷氨酰胺, 100 µM 2-基, LIF, 3 µMCHIR99021 和0.8 µM PD184352)。</li>
</ol>3. 延时显微镜
<ol><li>使用适合实时成像的显微镜, 允许控制温度和 CO2。将温度设置为37° c, CO2到5% 之前使用, 并允许平衡为 1-2 h。</li>
	<li>把盘子放到显微镜下, 找到适合于成像的斑点。选择那些单元格分布均匀但不太密集的斑点, 这将有助于分析。将所选点的数目调整为分析所需的单元格数。在这项研究中, 每细胞线记录 3-5 点。</li>
	<li>选择目标。20X 目标适合 ES 细胞的大小。</li>
	<li>选择要记录的荧光通道的光照设置。根据感兴趣细胞的荧光信号强度调整激光功率和曝光率。确保获得一个强大的初始信号, 因为它会减少在电影的过程中, 但避免激光功率高于 30%, 以尽量减少光和漂白。本研究采用 632/22 nm (波长/带通) 的励磁滤波器、679/34 nm (波长/带通) 的发射滤波器、10% 的激光功率和 100 ms 的曝光时间。</li>
	<li>选择时间推移实验的成像间隔和周期。对于预期半衰期为 2-20 小时的蛋白质, 选择 12-24 h 的捕获时间, 间隔为15分钟。为更短的居住的蛋白质, 减少间隔时间和承购时间, 为更长的居住的蛋白质相应地增加。</li>
	<li>开始成像。</li>
</ol>4. 图像处理与分析
<ol><li>以 tiff 格式将获取的图像作为堆栈收集。要进行进一步的处理和分析, 请使用斐济软件15。如果显微镜软件未将时间间隔数据收集成堆栈, 请在软件中打开时间推移实验的所有帧 (通过单击文件|打开...或将相应的文件拖放到工具栏上), 然后单击图像|堆栈|要堆叠的图像。</li>
	<li>使用减法背景函数从堆栈中的所有图片中删除背景。为此, 请单击进程|减去背景。在弹出窗口中选择滚动球半径。使用的半径至少是所成像单元格的大小。若要将背景减法应用于堆栈中的所有单元格, 请在 "进程堆栈" 弹出窗口中选择是。</li>
	<li>选择一个感兴趣的单元格, 并使用工具栏在它周围绘制一个感兴趣的区域 (ROI)。对于核信号, 使用椭圆形选择, 首先单击工具栏中相应的选项卡, 然后单击感兴趣的核心并调整其周围的椭圆。对于细胞质信号或非常密集的区域使用徒手选择可以密切遵循细胞轮廓。避免包含太大的背景区域, 即使没有必要精确地跟踪单元格的轮廓, 因为所有背景像素的后续减法 (步骤 4.8-4.9)。</li>
	<li>通过单击分析 |工具 |投资回报率经理 |在键盘上添加或按T 。</li>
	<li>通过在 "堆栈" 窗口中移动选项卡, 或按键盘上的 "shift + <", 然后重复前面的操作, 继续到下一帧。在整个电影过程中跟踪感兴趣的单元格, 并将每个帧的 roi 添加到 roi 管理器中。单击其他| 保存每个跟踪单元格的最终 ROI 集在 ROI 管理器中保存..。 注意: 在细胞分裂之后, 分开地跟踪两个子细胞并且在背景减法之后总结他们的综合强度 (参见步骤 4.6-4.9) 在细胞分裂期间考虑蛋白质稀释。保存两个子单元格的 ROI 集。</li>
	<li>在整个影片中跟踪感兴趣的单元格后, 单击度量, 以获取平均强度和 roi 区域的值。将获得的值复制到电子电子表格程序中, 并计算每个时间点的单元格的原始集成强度, 如下所示: <img alt="Equation 1" src="/files/ftp_upload/56604/56604eq1.jpg"> 其中平均值c是平均强度和区域c相应 ROI 的区域。</li>
	<li>若要估计本地背景, 请在每个时间框架的感兴趣的单元格附近添加一个 ROI。避免包括任何细胞荧光信号。使用一个近似于单元格大小的循环 ROI, 如果需要, 可以相应地移动或收缩它,例如如果相邻的单元格干扰。按照前面所述的蜂窝 roi 集进行操作, 以便获得背景 roi 集并将测量的强度值复制到电子表格中。</li>
	<li>为了获得对单元格的综合强度的背景校正值, 首先计算每个时间点的背景的综合强度: <img alt="Equation 2" src="/files/ftp_upload/56604/56604eq2.jpg"> 其中平均值BG 是背景信号的平均强度,区域C 是环绕单元格的 ROI 区域。不要使用背景 ROI 区域, 除非两个区域的大小相同。</li>
	<li>计算每个时间点的单元格的最终背景减去集成强度: <img alt="Equation 3" src="/files/ftp_upload/56604/56604eq3.jpg">
</li>
	<li>为了使单个细胞衰变曲线正常化, 将每个时间点的强度值除以第一个时间点的强度值。 注意: 曲线拟合 (参见步骤 4.11) 可以在每个单个单元格上执行, 也可在总体平均值上进行。如果计算基于总体的平均值以避免细胞间不同荧光强度的偏差, 则需要进行规范化。因此, 正常化确保每个单元对最终衰变曲线具有相同的权重。此外, 规范化可以很有用地可视化单个细胞衰变, 独立于其绝对荧光强度 (参见图 3 a-3 c)。但是, 如果只确定单单元半寿命, 且数据不平均, 则可以省略规范化步骤, 并可直接对原始数据执行曲线拟合。</li>
	<li>对于蛋白质半衰期的估计, 使用曲线拟合工具。在这项研究中, 使用了 matlab 曲线拟合工具箱 3.4.1, 它位于 matlab 用户界面的应用程序部分的默认位置。通过单击导入数据选项卡, 将电子表格中的荧光强度和时间值导入到 MATLAB 中. 打开 "曲线拟合工具箱", 然后在X 数据和Y 中选择时间点和荧光衰减数据数据选项卡. 选择自定义公式在 "曲线拟合" 选项卡中, 输入指数衰减的公式: <img alt="Equation 4" src="/files/ftp_upload/56604/56604eq4.jpg"> 其中, f (t)是给定时间点的荧光强度, a初始强度和b衰变率。在适应选项... 选项卡中, 为a和b的下限选择0。然后, a和b的估计值将显示在结果窗口中。计算半寿命如下: <img alt="Equation 5" src="/files/ftp_upload/56604/56604eq5.jpg">
</li>
</ol>

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作者没有什么可透露的。

当使用 SNAP 标签来监测蛋白质衰变时, 最关键的一步是确保在培养基中或在洗涤后的细胞中没有残留的未粘合染料, 否则它可能在实验过程中与新产生的 SNAP 标签分子结合在一起。伊破坏衰变曲线。这是一方面通过仔细执行所有描述的洗涤步骤实现的。另一方面, 染料的浓度应该保持尽可能低, 而仍然在一个范围, 允许获得一个良好的信噪比。如图 2所示, 应通过测试不同的稀释对每个实验的染料浓度进行优化。在我们的情况下, 强力霉素诱导细胞系具有较高的表达水平, 从而允许使用低染料浓度。
该协议描述了一种通过时间推移荧光成像来量化单细胞蛋白质降解的一般方法, 并且可以根据实验条件和相应的目标来修改协议的各个方面。实验.蛋白质降解主要遵循一级指数衰变。对于此处所示的蛋白质衰变, 拟合的 R2值相当高 (0.95-0. 99), 使用多指数并没有显著改善其适应度。然而, 如果从单指数拟合得到的结果不令人满意, 可考虑多指数, 这就意味着存在多个标记的蛋白质亚群以不同的速率衰减。此外, 我们的图像分析管道涉及大量的手工工作, 这是唯一可行的, 如果有限数量的细胞进行分析。对于更高数量的细胞, 应该考虑一种更自动化的方法。过去几年中, 各种自动化工具已经开发出来, 可以分割和跟踪较大数量的单元格16,17。然而, 细胞的移动和频繁的细胞分裂是挑战和容易导致分割错误。此外, 当成像蛋白衰变时, 荧光信号会随着时间的推移而减少, 使得分割阈值的选择更加困难。因此, 在执行此协议之前, 应仔细测试跟踪工具, 因为分割错误会使数据发生很大的扭曲。另外, 根据所获得的图像的质量, 可以考虑不同的背景减法方法。在这里, 我们使用斐济的滚动球校正, 这是适用于均匀光照图像与稀疏种子细胞。然而, 特别是如果图像遭受不均匀的光照, 更专业的背景减法可能会应用18,19。
我们的方法的一个局限是可以分析的时间推移录音的期限。为相对地短的居住的蛋白质, 承购时间大约 12 h 是充足的。为更长的居住的蛋白质, 作为例如 Srsf11, 执行曲线适应在 12 h 的时间范围是可能的。然而, 重要的是要记住, 指数适合可能是较不精确的较长寿命的蛋白质, 其采集时间和成像间隔应增加。如果快速循环细胞被成像, 较长的采集时间将使分析复杂化, 因为要分析的子细胞数量增加。同样, 在这种情况下, 更自动化的分析管道可能是有用的。此外, 在使用 snap 标记融合蛋白来确定蛋白质衰变率时, 应考虑对内源蛋白的半衰期进行改变的可能性。在这里使用的细胞线的情况下, 我们没有看到这样的效果的证据, 因为确定的半生命的 Oct4 和 Nanog 密切匹配已发布值3,20,21,22。为了进一步排除对蛋白质降解的影响, 特别是当没有关于半衰期的数据时, 一种选择是通过放线菌阻断蛋白质合成, 并使用抗体在不同的时间点进行西部杂交。对内源蛋白进行识别, 并直接比较标签内源蛋白的衰变。
大多数以前公布的蛋白质半衰期的数据是基于大量的生物化学实验, 主要是以时间为基础的西方印迹实验, 对蛋白质合成的抑制放线菌。但是, 这种方法不提供单个单元格的分辨率, 并且由于可以考虑的时间点有限, 所以其时间分辨率较低。相比之下, 我们的单细胞方法不需要使用蛋白质合成抑制剂, 并且可以识别蛋白质降解率中细胞的可变性。它还允许识别具有不同半衰期的细胞的潜在亚群, 这对异表达的蛋白质的研究可能特别有趣。我们的方法的另一个优点是在硅片中同步单元格并因此监视半衰期中的瞬态变化, 例如整个单元周期。但是, snap 标记技术需要生成相应的快照标记的单元格行, 这可能是其主要缺点。重要的是, 我们的方法也可以应用到内标记蛋白质9。如上所述, 我们没有观察到的任何证据的 snap 标签改变了内源性蛋白质半衰期的蛋白质, 我们已经研究, 建议的准确性的 snap 标签的方法是在同一范围内的其他方法。
这里介绍的方法对各种应用都很有用。最重要的是, 它提供了一个选项, 研究细胞对细胞的变化在半衰期的任何捕捉标签融合蛋白的兴趣。还有其他几个可用的标记 (如剪辑标记23或光晕标记24), 其工作原理是相同的。脉冲标记和洗涤残留染料将导致衰变曲线, 这允许确定蛋白质半衰期, 而离开染料在介质 (如果配体是非荧光, 除非绑定到标签), 使长期成像的蛋白质的利益。由于有大量的可供选择的荧光配体, 因此, SNAP 标签可以与其他标签或多个其他荧光标记分子结合在一起, 允许在不同的环境中应用该技术。

众所周知, 细胞蛋白具有广泛的周转, 合成和降解率是特定的每个蛋白质和受生理调节。传统上, 蛋白质降解率已被测量使用散装方法, 如放射性脉冲追逐分析, 或涉及蛋白质合成抑制剂, 如放线菌1。最近, 与质谱联用的细胞培养 (SILAC) 中的氨基酸的稳定同位素标记已经建立, 以在全球范围内量化蛋白质周转量2。但是, 这些方法受人口平均的限制, 因此, 关于细胞间可变性的信息就会丢失。此外, 无法确定在整个细胞中不同步的蛋白质降解的瞬态变化。
另外, 蛋白质半衰期也可以通过荧光的方法来确定, 这通常具有提供单细胞分辨率的优点。例如, 一个 photoactivatable 的绿色荧光蛋白 (paGFP) 已经被用来确定早期哺乳动物胚胎中的 Oct4 半衰期3。另一种监测活细胞中蛋白质衰变的方法是使用捕捉标记与荧光延时成像相结合。SNAP 标签是一个变种版本的 dna 修复酶 O6-alkylguanine dna-alkyltransferase (AGT), 特别是与 benzylguanine (BG) 衍生物, 这可以耦合到分子探针4,5,6. 因此, 任何捕捉标签的融合蛋白都可以用荧光、细胞渗透染料进行不可逆转的标记。脉冲标记的兴趣蛋白融合到 SNAP 标签, 其次是残留染料的冲洗, 允许监测的标记蛋白的人口的退化, 从而确定蛋白质半生命。SNAP 标签已成功地用于蛋白质的脉冲追逐标记和确定蛋白半生命的黏附细胞培养和在体内5,7,8,9。在商业上有大量的对齐标签衬底覆盖常用的荧光光谱, 使每个特定应用的最佳染料的选择。因此, SNAP 标签也可以用于多色成像结合其他荧光融合蛋白或染料。细胞不渗透性染料适用于膜栓蛋白的标记, 而细胞通透性染料则适用于细胞内和膜结合蛋白的监测。此外, 这些探针中的一些几乎没有基本的荧光, 只有在绑定到 SNAP 标记10时才开始发出强烈的荧光信号。
本协议描述了如何使用 SNAP 标签测量单个细胞中不同蛋白质的降解率。我们将这种方法应用于小鼠胚胎干细胞培养的 e-钙黏蛋白, 但它应该有可能使用它与任何黏附培养的细胞类型。我们表明, 脉冲标记的快照标签融合蛋白, 其次是荧光时间推移成像, 允许确定的单个细胞半衰期的各种蛋白质的兴趣, 并提供了一个估计的细胞变化的一半生命在培养细胞的数量。

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Equipment</td>
			<td style="width:189px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">ES cell line expressing a SNAP-tag fusion protein of interest</td>
			<td style="width:189px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 100 mm TC-Treated Cell Culture Dish</td>
			<td style="width:189px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">96 Well, Black/Clear, Tissue Culture Treated Plate</td>
			<td style="width:189px;">Corning</td>
			<td style="width:119px;">353219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Neubauer-improved counting chamber, 0.1 mm</td>
			<td style="width:189px;">Marienfeld-superior</td>
			<td style="width:119px;">640030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">CO2 Incubator</td>
			<td style="width:189px;">Panasonic</td>
			<td style="width:119px;">MCO-170AICUV-PE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Centrifuge 5804 R</td>
			<td style="width:189px;">Eppendorf</td>
			<td>5804000528</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">InCell Analyzer 2200 Cell Imaging System</td>
			<td style="width:189px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">29027886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name</td>
			<td style="width:189px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Reagents</td>
			<td style="width:189px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Glasgow Minimum Essential Medium</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td>G5154</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Fetal Bovine Serum, embyonic stem cell-qualified</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">16141-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Sodium pyruvate solution</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">113-24-6</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Minimum Essential Medium&#160; Non-Essential Amino Acids</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">11140-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin</td>
			<td style="width:189px;">BioConcept</td>
			<td style="width:119px;">4-01F00H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-Glutamine 200mM</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2-Mercaptoethanol</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">63689-25ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Leukemia Inhibitory factor</td>
			<td style="width:189px;">-</td>
			<td style="width:119px;">-</td>
			<td>Produced in the lab by transient transfection of HEK-293T cells, followed by collection and filtering of the supernatant.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CHIR99021 (GSK-3 Inhibitor XVI)</td>
			<td style="width:189px;">Merck Millipore</td>
			<td style="width:119px;">361559</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">PD 0325901</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">391210-10-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Gelatin from bovine skin</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">9000-70-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#39;s PBS 10x concentrated</td>
			<td style="width:189px;">BioConcept</td>
			<td style="width:119px;">3-05K00-I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#39;s PBS Without Ca++/Mg++</td>
			<td style="width:189px;">BioConcept</td>
			<td style="width:119px;">3-05F29-I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Trypsin-EDTA-Solution 0.25%</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Recombinant Mouse E-Cadherin Fc Chimera protein</td>
			<td style="width:189px;">R&#38;D systems</td>
			<td style="width:119px;">748-EC-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Doxycycline hyclate</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SNAP-Cell 647-SiR</td>
			<td style="width:189px;">New England BioLabs</td>
			<td style="width:119px;">S9102S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">FluoroBrite DMEM</td>
			<td style="width:189px;">ThermoFisher</td>
			<td style="width:119px;">A18967-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name</td>
			<td style="width:189px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Software</td>
			<td style="width:189px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">FIJI</td>
			<td style="width:189px;">-</td>
			<td style="width:119px;">-</td>
			<td>Open-source image analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">MATLAB R2014a</td>
			<td style="width:189px;">Mathworks</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Microsoft Excel</td>
			<td style="width:189px;">Microsoft</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell quantification of protein degradation rates by time-lapse fluorescence microscopy in adherent cell culture

蛋白质处于合成和降解的动态状态, 它们的半衰期可以在各种情况下进行调节。然而, 最常用的测定蛋白质半衰期的方法要么仅限于裂解细胞的总体平均值, 要么需要使用蛋白质合成抑制剂。该协议描述了一种方法来测量蛋白质半衰期在单一的生活粘附细胞, 使用捕捉标签融合蛋白结合荧光时间推移显微镜。任何有兴趣的蛋白质都可以通过与 benzylguanine 衍生物结合的荧光、细胞渗透染料与一个标签的共价键结合, 并且在残留染料被冲蚀后可以对标记蛋白的衰变进行监测。随着时间的推移, 细胞的跟踪和综合荧光强度的量化导致了每个跟踪细胞的指数衰减曲线, 从而可以通过曲线拟合确定单个细胞的蛋白质降解率。这种方法为培养细胞中半生命体的异质性提供了一个估计, 这是不能用其他方法来评估的。这里提出的方法适用于任何类型的培养粘附细胞表达的兴趣蛋白融合到一个 SNAP 标签。在这里, 我们使用小鼠胚胎干细胞生长在 e-钙黏附素涂层细胞培养板, 以说明如何单细胞降解率的蛋白质与广泛的半衰期可以确定。

所描述的协议提供了一个估计的细胞的可变性在半衰期的任何给定的蛋白质融合到一个 SNAP 标签。使用重组 e-钙黏附素-Fc 用于涂层的成像板允许单细胞分辨率在 ES 细胞, 否则生长在殖民地。可以在整个影片过程中单独跟踪单个单元格 (图 1A)。
为了确定每个单细胞的蛋白质半衰期, 重要的是要测量的集成, 背景扣除的 SNAP 标签荧光信号随着时间的推移 (图 1B), 总结了两个子细胞的综合强度案件的分歧。这在一个指数衰减曲线为每个细胞, 衰变率和因而半生活可以由曲线拟合提取 (图 1C)。重要的是, 如果计算出平均衰变曲线, 则应将单个单元格的轨迹归一化到第一帧, 以确保每个单元的平均重量相同, 尽管在初始荧光强度之间存在不同的细胞。
适当的染料浓度取决于所使用的染料和细胞线的类型, 因此应对每个试验进行优化。为了说明这一点,图 2显示了用于所有实验的远红光荧光基底的不同浓度的衰变曲线, 在强力霉素诱导的捕捉标记融合细胞线上进行了测试。根据制造商的说明选择了3µM 的初始染料浓度, 并对1:3 进行了连续稀释, 直到达到 1.4 nM 的浓度。对于两个最高浓度的信号不减少, 而观察衰变指数为浓度低于 111 nM。选择 12 nM 染料的最佳浓度进行进一步的实验, 因为它保证了在洗涤后的培养基中残留的少量染料, 但信号仍然足够亮, 可以观察到多种强力霉素诱导的明显的衰变曲线。细胞系。
图 3 3 c 显示代表性结果, 包括单细胞衰变和人口平均值, 为3蛋白质以不同的平均半衰期: 多伴生的转录因子 Nanog (平均半寿命 2.9 h) 和 Oct4 (平均半衰期: 4.8 h), 和更长的活前 mRNA 处理因子 Srsf11 (平均半衰期 25.3 h)。boxplots (图 3D) 代表了通过曲线拟合从单个细胞衰变曲线获得的半衰期, 并说明了三种蛋白质的半生命的异质性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56604/56604fig1.jpg"> 图 1: 时间间隔获取和分析 答: 一系列具有代表性的图像, 显示荧光在细胞脉冲标记的染料的衰变。细胞系: 强力霉素诱导的 TRE3G-Oct4-SNAPtag, 诱导与 500 ng/毫升强力霉素7小时前成像。 B: 电影综合强度的分析和计算。ic: 集成强度的单元格, ibg: 综合强度的背景, ic bg: 背景校正综合强度。 C: 用 MATLAB 进行指数曲线拟合。一个单细胞衰变迹的例子, 相应的拟合和结果估计的衰变参数 a 和 b。半寿命计算如下: t1/2 = ln (2)/b。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56604/56604fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56604/56604fig2.jpg"> 图 2: 染料浓度的优化. 荧光衰变的细胞脉冲标记不同浓度的对齐染料。从3µM 开始的染料的连续稀释被执行了。这些痕迹平均每5细胞, 正常化到第一帧和跟踪 12 h. 细胞线: 强力霉素诱导 TRE3G-sfGFP-mOrange2-SNAPtag-PEST, 诱导与 500 ng/毫升强力霉在成像前24小时。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56604/56604fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56604/56604fig3.jpg"> 图 3: 对不同的快照标记候选蛋白质的半衰期为单细胞可变性. A-c: 单细胞衰变 (浅蓝) 和人口平均 (深蓝色) 的强力霉素诱导 Nanog, Oct4 和 Srsf11-SNAP-tag 融合蛋白。 D: 单个细胞的半衰期, 这是由每个单细胞的指数曲线拟合计算的。boxplots 显示在日志2比例中。方框显示分范围 (IQR), 黑线表示中间值。晶须代表最小和最大值, 不包括离群 (黑点), 这是定义的值偏离超过1.5x 的 IQR。N = 20 单元格。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56604/56604fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture at JoVE.com

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

该协议描述了一种方法, 以确定蛋白质半生命的单一生活粘附细胞, 使用脉冲标记和荧光时间推移成像的捕捉标签融合蛋白。

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

single-cell-quantification-protein-degradation-rates-time-lapse

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.5K 次观看.  École Polytechnique Fédérale de Lausanne (EPFL). 该协议描述了一种方法, 以确定蛋白质半生命的单一生活粘附细胞, 使用脉冲标记和荧光时间推移成像的捕捉标签融合蛋白。

视频: Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...