我们感谢 (Ulmschneider 对《议定书》的贡献和戴安娜理发师对手稿的建议。这项工作由国家卫生研究所资助, GM116384 T.G. Nystul 和 d.l 理发。

注意: 要测量在 FSC 谱系中的φ, 我们计算在生理条件下演、全氟和卵泡细胞中 pHluorin 的荧光强度与 mCherry 的比值, 并将比值转换为φ值和标准每个单元格类型 7 的校准曲线。首先, 进行活体成像实验, 测量 germaria pHluorin 和 mCherry 的荧光强度, 将其分解为包含 NaHCO 3 的缓冲区, 模拟生理条件 1 , 7 . 接下来, 通过在 Na + 中测量 pHluorin 和 mCherry 荧光强度生成标准曲线, 其中包含载体尼日利亚调整为两个不同的 pH 值, 6.5 和7.5 的 K + 缓冲区。在尼日利亚的存在下, φ均衡与细胞膜上的缓冲液的 ph 值相匹配, 导致皮与胞外 ph 值匹配。最后, 标准曲线用于将 pHluorin 与 mCherry 比值转换为估计的φ值. 
 1. 预试: 在活体上测量前的准备 注: 要测量φ在体内的 , mCherry::p hluorin 转基因必须表达在细胞类型的兴趣。下面是在 FSC 谱系中产生转基因 pHluorin 蝇的一些常用方法。生成 mCherry::p hluorin 克隆特别适用于识别演, 后者位于 FSC 克隆的前边缘。组织特异性 mCherry::p hluorin 表达是有用的, 以测量皮横跨整个组织, 也更方便, 当与表达的 rna 干扰或转基因. 
<ol> <li> 转基因 pHluorin 的生成 <ol> <li> mCherry::p hluorin 标记的 fsc 克隆 <ol> <li> 使 mCherry::p hluorin fsc 克隆, 交叉无人攻击-mCherry::p hluorin 1 Act-Gal4 flipout 的股票或其他股票与 Flp/首次登记税诱导 Gal4. </li>
			<li> 使 heatshock 诱导克隆, heatshock 成人 F1s 2 天后羽化在空瓶中为1小时在一个37和 #176; C 水浴, 四次大约每 12 h. </li>
			<li> 为了确保在这一期间的正常生长和最大卵子率, 在克隆诱导后至少6天内每天提供新鲜的湿酵母 (大约相等的部分干面包和 #39 的酵母和水) 保持苍蝇. </li>
		</ol> </li> <li> 组织特定 mCherry::p hluorin 表达式 </li> <li> 交叉无人机-mCherry::p hluorin 1 到卵泡细胞特定的驱动程序, y 1 w *;P{GawB}10930/CyO (布卢明顿 ID: 7023). </li>
		<li> 3 天后, 苍蝇开始 eclosing, 收集和放置在含有湿酵母的小瓶, 至少24小时前解剖. </li>
	</ol> </li> <li> 制作解决方案 注意: 在实验当天准备下列缓冲器很重要, 因为缓冲液中的 pH 值和溶质浓度会随着时间的推移而变化。
	<ol> <li> 准备碳酸氢盐、尼日利亚和夹层缓冲。按列出的顺序添加组件以避免沉淀物的形成 (请参阅 表 1 、 表 2 和 表 3 ). </li>
	</ol> </li> </ol> 2。试用: 在 FSC 谱系中测量φ 注意: 对碳酸氢盐缓冲液和两个尼日利亚缓冲条件的解剖、安装和现场成像步骤可能需要执行两次: 一次确定显微镜设置和第二次收集实验数据。有关详细信息, 请参阅下面的2.2 节. 
<ol> <li> 剥离和安装 注意: 对于执行此步骤所需的材料, 请参阅 图 1 和 材料表 。对于3D 打印的安装箱, 3D 打印机文件作为 补充文件 1 和 补充文件 2 提供。
	<ol> <li> 剥离: <ol> <li> 通过将一层薄薄的真空油脂涂敷到3D 打印安装腔的平坦一侧来准备安装箱。放置在一个 22 x 40 毫米片的一侧, 以密封片的安装室. </li>
		</ol> </li> <li> 在500和 #181 中解剖雌性成虫的卵巢; 解剖缓冲液 (重碳酸盐或尼日利亚缓冲, 表 3 ) <ol> <li> 麻醉 3-4 苍蝇使用 co 2 气体, 并将苍蝇转移到飞垫上的 co 2 气体. </li>
			<li> 拿起一只带钳子的麻醉苍蝇, 放入解剖介质. </li>
			<li> 用一个 cep 捏住苍蝇的胸腔, 在它的腹部的尖端用力拉扯, 直到它的卵巢暴露出来. </li>
			<li> 在从其他器官分离卵巢后, 用 22 1/2 注射器针将 ovarioles 分开。仔细分离的肌肉鞘从卵巢和单独的个体 ovarioles 从 ovarioles 群。一种有效的分离肌鞘的方法是用一根注射器将 ovarioles 的簇放在其前端, 并沿着单 ovarioles 的长度向下划. </li>
			<li> 在解剖介质中孵育解剖的卵巢10分钟, 然后再进行安装步骤, 以便有足够的时间让φ细胞完全平衡尼日利亚解剖介质的 pH 值. 
			注意: 确保从 ovarioles 中取出肌肉鞘。肌肉鞘可能衰减荧光信号, 这使得很难识别单个细胞使用 Concanavilin A, 并可能导致 ovarioles 自发移动的实时成像. </li>
		</ol> </li> <li> 安装 <ol> <li> 将一小滴解剖介质放在安装腔内的玻璃片上。
			<li> 使用镊子转移分离的 ovarioles. </li>
			<li> 分离后阶段的蛋室从 germaria 与早期阶段的蛋室, 并试图确保解剖 germaria 被放置在解剖媒体下降的中心. </li>
			<li> 在12毫米片的两侧添加两滴指甲油, 让它在大约十年代风干. </li>
			<li> 将圆形片放在包含分离 germaria 的解剖介质上, 使该侧与指甲油朝下并接触解剖介质。按下的边缘部分与指甲油铺平和确保 germaria 的位置。我们建议使用较旧的指甲油, 因为它在片表面上的涂抹较少, 因为它在固定到位. </li>
			<li> 用额外的解剖介质填充安装腔。不包含豆的条件特定缓冲区-一种染料可以用来填充腔室. 
			注: 解剖和安装所用的总时间不应超过15分钟。这是很重要的, 以减少组织损伤的影响和细胞死亡. </li>
		</ol> </li> </ol> </li> <li> 实时成像: 注意: 在此步骤中, 首先从碳酸盐条件和两个尼日利亚条件中收集图像, 以确定适当的显微镜设置; 根据需要调整显微镜设置,然后收集实验数据的图像。使用共聚焦显微镜能够成像3通道: GFP (475 excitation/509 发射), mCherry (575 excitation/610 发射), 和远红色 (633 excitation/647 排放). 
	注: 设置显微镜设置: 收集的图像的像素强度将被量化以确定φ的估计值, 因此, 每个图像集中的信号的强度数值既不太低也不饱和, 这一点很重要。可以在图像中捕获的强度范围由图像位深度定义。在许多情况下, 默认情况下会生成8位图像, 但我们建议将这些数据作为16位图像进行获取, 这有一个更广泛的动态范围。重要的是要确保在荧光通道之间的串扰最小化, 因此设置应调整, 以便从每个荧光收集的发射频谱不重叠。若要测试这些设置, 请使用 GFP 的激发频谱采样图像, 并在 mCherry 和 反之亦然 的发射频谱中收集。如果设置已正确调整, 则两种情况下都不应出现信号。设置显微镜设置 ( 即 , 激光扫描共焦的电压设置, 激光功率和针孔尺寸), 使所有三图像集中的信号的像素强度都在图像文件的动态范围内。在我们所有的实验中, 我们使用了一个40x 目标的白光激光扫描共聚焦显微镜, 以获得16位图像的1024和 #215; 1024 格式, 同时我们根据需要优化每个实验的电压增益。例如, 在一个实验中, 我们分别将 GFP、mCherry 和远红的电压增益设置为37.8%、72.9% 和260%。
	<ol> <li> 图像 ovarioles 在尼日利亚解剖缓冲区的 pH 值为 6.5, 并调整设置, 使 pHluorin 和 mCherry 图像的像素强度较低, 但不低于检测的相机的限制. </li>
		<li> 图像的另一组 ovarioles 在尼日利亚解剖缓冲区在 pH 值 7.5, 并调整设置, 使 pHluorin 和 mCherry 图像的像素强度高, 但不饱和. </li>
		<li> 在碳酸氢钠夹层的图像卵巢缓冲, 并确保 pHluorin 和 mCherry 图像的像素强度不饱和的选择设置。如果像素已饱和, 则根据需要调整设置. 
		注: 显微镜设置参数可能因所使用的显微镜设置而异, 并且必须对每个实验进行优化。在显微镜设置之后, 不要在实验的其余部分改变它们。设置应在控制和实验条件之间保持一致。在我们最初的皮实验中, 我们生成了2和3点尼日利亚校准曲线, 并发现用这两种方法计算的φ没有显著差异。但是, 一般情况下, 在校准曲线中添加更多的点将有望提高精确度。因此, 我们建议生成具有不同点数的曲线, 以确定给定的一组实验条件需要多少. </li>
	</ol> </li> <li> 数据收集: <ol> <li> 在碳酸氢盐和尼日利亚缓冲条件下对样品进行解剖、安装和成像。经过解剖和安装, 最大时间花成像一组样本不应超过45分钟, 以确保荧光强度测量是在活的, 健康的组织. </li>
		<li> 对于每个条件, 获取至少 5 germaria 的图像. </li>
	</ol> </li> </ol> 3。后试用: 图像分析 <ol> <li> 测量演、全氟和卵泡细胞中的荧光强度 <ol> <li> 背景减法: <ol> <li> 在单独的窗口中每个通道打开未处理的图像 ( 图 4C ). </li>
			<li> 使用矩形工具在图像的一部分中绘制 pHluorin 通道窗口中的感兴趣区域 (ROI), 而不显示信号. </li>
			<li> 可选步骤: 设置阈值的下限, 以便排除具有背景下强度值的像素, 并将阈值的上限设置为最大。当以这种方式设置阈值时, 没有信号的图像区域大多为蓝色, 并且信号清晰可见 ( 图 4A ). </li>
			<li> 测量 ROI 中的平均荧光强度。如果阈值是在步骤3中设置的, 请确保在 "设置度量值" 对话框中检查和 #34; 限制为阈和 #34; 框. </li>
			<li> 通过在进程和 #8594 中找到的减法函数减去图像的每个通道和切片的实测背景强度; 数学菜单. </li>
			<li> 对 mCherry 通道窗口重复步骤 3.1. 1.2-6, 但在步骤3.1.1.2 中, 不要使用矩形工具绘制新的矩形, 而是在 "编辑与 #8594" 中找到 "还原选择" 函数, 并在 "选择" 菜单中添加一个矩形。图像上的尺寸和位置. </li>
		</ol> </li> <li> 标识演、全氟和卵泡细胞: <ol> <li> 通过使用豆的形态学和位置来识别演、全氟和卵泡细胞--一种染色来查找细胞边界 ( 图 2 ). </li>
			<li> 演是位于 germarium 区域 2 a/2 b 边界的边缘的薄三角形单元格。如果 mCherry::p hluorin 是在 fsc 克隆中表达的, 那么 fsc 也可以被确定为克隆的前多数细胞. </li>
			<li> 全氟化是区域2b 中的不规则形状的细胞, 紧邻演和下游. </li>
			<li> 卵泡细胞是正方形或柱状细胞, 周围的生殖细胞囊肿3区. </li>
		</ol> </li> <li> 获取荧光强度比 <ol> <li> 选择一个或多个切片, 其中感兴趣的单元格在 mCherry 通道中具有最高的荧光强度, 并确保豆--在远红色通道中看到的染色是在矢量我们在选定的切片. </li>
			<li> 在每个 FSC 的周围绘制 ROI, 并测量所有选择用于测量的切片中的 pHluorin 和 mCherry 的平均荧光强度. </li>
			<li> 通过 mCherry 通道的平均荧光强度除以 pHluorin 通道的平均荧光强度来计算 pHluorin 与 mCherry 荧光的比值. </li>
		</ol> </li> </ol> </li> <li> 派生φ值并生成 pseudocolored 图像 <ol> <li> 导出φ值 <ol> <li> 在所有情况下, 应使用相同基因型的果蝇卵巢生成线性回归曲线实验和控制条件。使用两个尼日利亚缓冲区条件 ( 图 3 ) 中的数据为每个单元格类型生成线性回归曲线。在 Excel 中, 可以通过在图表上绘制数据并添加线性趋势线来完成此操作。或者, 请参见: </li> <li> 使用从尼日利亚条件和直线 (y = mx + b) 计算的线性回归曲线的斜率和 y 轴截距, 将 pHluorin 转换为从碳酸盐样品中计算出的 mCherry 比值值。条件 pH 值。在这个等式中, 将斜率形式 (y 截距) 替换为 b, 将比值值放入 x, 并求解 y. </li>
		</ol> </li> <li> 生成反映斐济的比率值的 pseudocolored 图像。
		<ol> <li> 按照上面的过程进行背景减法 (步骤3。1.1 和 图 4 ). </li>
			<li> 使用 "过程" 菜单中的 "图像计算器" 功能将 pHluorin 通道从 mCherry 通道中分离出来。确保 #34; 创建新的窗口和 #34; #34; 32 位 (浮点) 结果和 #34; 复选框都已选中。更改查找表 (在 "图像" 菜单下找到), 将其转换为 "热" 或 "选择"。有关其他合适的选项, 请参见 图 4 . </li>
			<li> 在 "调整亮度和对比度" 对话框 (图像和 #8594; 调整和 #8594; 亮度/对比度) 中, 单击和 #34; 设置和 #34; 按钮。将最小显示值设置为 0, 最大显示值为最能捕获数据的比率, 通常为 1.0-2.0 ( 图 5 ). </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+signaling+promotes+self-renewal+through+the+establishment+of+cell+polarity+in+Drosophila+follicle+stem+cells.">EGFR signaling promotes self-renewal through the establishment of cell polarity in Drosophila follicle stem cells.</a> eLife. 3, (2014).</li><li>Margolis, J., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+behavior+of+epithelial+stem+cells+in+the+Drosophila+ovary.">Identification and behavior of epithelial stem cells in the Drosophila ovary.</a> Development. 121 (11), 3797-3807 (1995).</li><li>Nystul, T. G., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epithelial+niche+in+the+Drosophila+ovary+undergoes+long-range+stem+cell+replacement.">An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement.</a> Cell stem cell. 1 (3), 277-285 (2007).</li><li>Morris, L. X., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live+imaging+provides+new+insight+into+stem+cell+regulation+and+germline-soma+coordination+in+the+Drosophila+ovary.">Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary.</a> Development. 138 (11), 2207-2215 (2011).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends in neuro. 24 (5), 251-254 (2001).</li></ol>

作者没有什么可透露的。

在这里, 我们描述了一种测量野生组织中的 FSC 谱系中的细胞 pHi 的方法。自从我们开始研究皮在果蝇卵巢组织中的应用以来, 这项协议已经在过去的五年中得到了发展和完善。在此期间, 该协议已成功地应用于我们的实验室和至少四不同的旋转光盘和激光扫描显微镜的多个调查人员。我们最初的观察的重现性, φ增加作为细胞在 FSC 谱系区分从干细胞到 pFC 到卵泡细胞状态, 横跨这些多次试验表明这生物现象是健壮的, 并且这种方法是可靠的。然而, 在我们的经验中, 这是一个具有挑战性的程序来掌握。它需要密切关注每一个步骤的细节, 并迅速, 高度熟练的解剖, 安装和成像步骤执行。如议定书所述, 解剖和安装程序包括一个10分钟的孵化, 但不应超过15分钟的总和和成像程序必须完成45分钟。在理想条件下,果蝇ovarioles 可以在 14 h17的区域性中进行维护, 但我们发现, 在用于生成标准曲线的尼日利亚缓冲区中, 组织开始更快地死亡。我们的指导方针确保所有的数据收集, 而组织仍然是在时间的窗口, 它看起来健康和形态正常。不过, 这并不会给每个步骤留下多少时间, 因此, 提前计划以确保一切都准备就绪, 从一个步骤到下一步是很重要的。事实上, 如果两个人在解剖、安装和成像过程中一起工作, 那就更有效率, 一个人执行一个步骤, 而另一个人准备下一步。
由于 phi 可能是敏感的实验操作所需的图像组织活体, 如温度和缓冲成分, 这种方法是最好的确定相对变化 phi。由于探针由两种不同的荧光蛋白组成, 它们的荧光特性如量子产率、生命时间和折叠特性可能会因各种细胞类型而受到不同程度的影响。因此, 必须包括尼日利亚缓冲条件处理的样品, 并使用它们在每一个试验中为每个细胞类型生成一个新的校准曲线。为了尽量减少额外的可变性来源, 强调了在成像过程中保持所有条件的一致性。尼日利亚缓冲条件下的样品应在重碳酸盐缓冲条件的同一天制备和成像, 所有样品的制备和图像采集应尽可能保持一致。这是很重要的, 因为 phi 测量是基于图像集之间的比较, 所以实验差异, 影响检测的 mCherry 和 pHluorin 在一个或多个图像集中可能会降低 phi 估计的准确性。诸如光电倍增管电压设置的变化 (如果使用激光扫描共聚焦) 或曝光时间 (如果使用旋转圆盘共焦) 或肮脏的镜头, 减少光线到达相机的数量, 将明显影响结果.但也有许多其他的微妙因素, 可能从日常变化, 也可能影响的结果, 例如, 如果苍蝇是营养充足, 苍蝇的年龄, 以及多久激光器已经在成像之前。准备和成像在同一天的所有三条件最小化这些差异, 从而产生最一致的结果。值得注意的是, 每次我们切换到一个新的显微镜或显微镜的组成部分升级, 有必要优化新设备的设置。这通常会导致个别测量的平均值发生变化, 但是, 只要每次试验产生新的校准曲线, 设备的变化对 pHi 估计值的影响极小。此外, 相关的比较通常是在同一组织内的不同细胞类型之间 (例如, 如、演、全氟和卵泡细胞), 从而在内部控制实验变异。
虽然本议定书的重点是测量皮野生组织, 该方法是兼容的标准果蝇方法的操纵基因表达, 如表达的 rna 干扰或转基因使用 UAS/Gal4, 和马赛克分析。自 mCherry::p hluorin 是由一个无人机启动器驱动, 它将始终是 co-expressed 与 rna 干扰或转基因, 和 MARCM18可用于产生与 mCherry::p hluorin 作为克隆标记的纯合突变体克隆。基于上述原因, 野生组织应被分析为一个控制和每一个突变基因型的试验。最后, 这里描述的一般原则可以用于 mCherry 的使用::p hluorin 在其他组织中测量皮。实际上, 该协议是从使用 mCherry 的协议改编而来的::p hluorin 在果蝇眼7中测量φ, 我们使用了类似的方法来图像 mCherry: 在幼虫脑中:p hluorin。总的来说, 这里描述的工具和方法提供了新的机会, 以研究在活体和完整的组织内, pHi在体内的多种功能。

最近的研究揭示了一个角色的变化在 pHi 细胞分化和发育不良在体内1,2。这些研究发现, 在同一类型的细胞中, pHi 在相同的分化阶段是非常一致的, 但它随着细胞从一个阶段过渡到另一个。在某些情况下, 阻断皮下的改变会部分地破坏分化, 这表明 phi 的变化不仅是细胞命运改变的结果, 反而有助于促进细胞命运的改变, 也许通过对 pH 敏感的调控作用分化所需的蛋白质或化学反应。未来的研究有可能揭示更多的洞察力的许多不同的作用, pHi 动力学在体内。然而, 研究 phi 在分化过程中的一个挑战是在体内获得精确的φ测量。与其他分化特征不同, 如细胞形态学和基因表达的变化, φ是细胞的不稳定的化学性质, 是不保存在细胞, 已固定和化的标准方法。此外, pHi 可能不稳定的细胞, 是压力或死亡的实验操作的结果。因此, 在测量 pHi 时保持细胞存活和尽可能健康是很重要的。有几种重要的染料可用于在培养基中测量细胞的φ3, 但在许多情况下, 它们不适合用于活体研究, 因为它们不能深入或均匀地穿透组织以提供精确的测量.
为了绕过染料渗透不良的问题, 我们和其他人使用了一个基因编码探针, mCherry::p hluorin4,5,6,7, 可以在单元格中明确表示类型的兴趣和影像活组织。pHluorin 是一个变种的 GFP 与较高的 pKa (〜 7.0 vs. 4.0), 折叠更容易在较高的 pH 值;因此, 细胞中 pHluorin 分子的总荧光强度随着φ8的增加而增加。重要的是, 荧光在皮值的正常胞浆范围内是线性的。相比之下, mCherry (pKa ~ 4.5) 的荧光对胞浆范围内的 pH 变化不敏感。这两个记者是共用一个单一的嵌合蛋白, 由单一的开放阅读框架编码, 所以他们总是在同等数量的存在。因此, pHluorin 与 mCherry 荧光强度的比值提供了对每个细胞中的探针浓度进行归一化的φ的测量。然后, 该比值可以转换为φ值的估计使用一个标准的曲线, 这是产生的 pHluorin 的 mCherry 比例从组织已经平衡已知的 pH 值。
在这里, 我们描述了使用 mCherry 的方法::p hluorin 测量的上皮 FSC 谱系的皮在果蝇卵巢。该良好组织已被用于模型的许多不同方面的上皮生物学, 如干细胞自我更新和分化9,10,11, 集体细胞迁移12, 以及单元极性的开发和维护13,14。卵泡上皮是由两个演产生的, 它们驻留在组织的前边缘, 结构称为 germarium15,16。这些细胞在成年期间定期分裂, 以自我和产生后代, 称为 prefollicle 细胞 (全氟化), 可以重新进入利基, 成为一个 FSC 或分化成三种不同的卵泡细胞类型: 极性细胞, 茎细胞, 或主体卵泡细胞。我们先前显示, 在野生组织中, phi 在分化的早期阶段稳步增加, 从演的φ6.8 到7.0 的全氟化合物, 到7.3 的卵泡细胞2。通过干扰无所不在表达的钠/质子交换器, DNhe2, 阻止这种增加, 严重损害了 pFC 的分化, 而通过过度表现的DNhe2导致轻度过量分化型.这些结果表明, pHi 是稳定地保持在早期 FSC 谱系, 它可以实验增加或减少在体内。这里描述的方法可以用来测量φ在野生组织或各种形式的突变组织, 包括 rna 干扰或过度表达使用 Gal4 的兴趣, 和有丝分裂克隆。

<table>
	<tbody>
		<tr>
			<td>Fly Stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-mCherry::pHluorin[1]</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>y1 w*;P{GawB}10930/CyO</td>
			<td>Bloomington Stock Center</td>
			<td>7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Act-Gal4 flipout stock</td>
			<td>Bloomington Stock Center</td>
			<td>4409</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals for Buffer preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P-3911</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Mallinckrodt</td>
			<td>4912</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP310</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Thermo Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C-5080</td>
			<td></td>
		</tr>
		<tr>
			<td>HCO3</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M-9272</td>
			<td></td>
		</tr>
		<tr>
			<td>NMDG+</td>
			<td>Sigma Aldrich</td>
			<td>M-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Mallinckrodt</td>
			<td>7088</td>
			<td>Use to Make KHPO4 pH 7.4</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP362</td>
			<td>Use to Make KHPO4 pH 7.4</td>
		</tr>
		<tr>
			<td>Concanavalin A, Alexa Fluor 647 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>C21421</td>
			<td>0.25 mg/ml dilution</td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Thermo Fisher Scientific</td>
			<td>N1495</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dissection and mounting tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 Dumont Inox forceps (Size 5)</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9473431</td>
			<td></td>
		</tr>
		<tr>
			<td>2 23-gauge syringe needles</td>
			<td>Sigma Aldrich</td>
			<td>Z192457</td>
			<td></td>
		</tr>
		<tr>
			<td>9-well glass dissecting dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-748B</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Grease</td>
			<td>Dow Corning</td>
			<td>1018817</td>
			<td></td>
		</tr>
		<tr>
			<td>22 X 40 mM glass coverslips</td>
			<td>Thermo Fisher Scientific</td>
			<td>12545C</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Glass Coverslips, 12mm diameter, 0.13-0.16mm thickness</td>
			<td>Ted Pella, Inc.</td>
			<td>26023</td>
			<td></td>
		</tr>
		<tr>
			<td>3-D mounting chamber</td>
			<td>custom manufactured</td>
			<td></td>
			<td>.stl and .ipt files for 3-D printer included as supplemental files</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-620-183A</td>
			<td>Model: Accumet AB15</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Olympus Corporation</td>
			<td>0H11436</td>
			<td>Model: SZ2-ST</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>SP5 or SP8 laser-scanning confocal microscope with a 40&#215; objective with a numerical aperture of 1.3</td>
		</tr>
	</tbody>
</table>

methods for imaging intracellular ph of the follicle stem cell lineage in live drosophila ovarian tissue

细胞内 pH 值 (pHi) 的变化在调节许多细胞功能, 包括新陈代谢、增殖和分化方面起着重要作用。通常, phi 动力学是确定的培养细胞, 这是经得起测量和实验操纵 phi。然而, 最近的新工具和方法的发展使得有可能研究皮动力学在完整的, 活组织。对于果蝇的研究, 一个重要的发展是产生一个携带 pHi 生物传感器的转基因线, mCherry::p hluorin。在这里, 我们描述了一个协议, 我们经常使用的成像直播果蝇ovarioles 在 mCherry 的上皮卵泡干细胞 (FSC) 谱系中测量φ::p hluorin 转基因野生型品系;然而, 这里描述的方法可以很容易地适应其他组织, 包括翼盘和眼上皮。我们描述的技术表达 mCherry::p hluorin 在 FSC 的血统, 维持卵巢组织在现场成像, 并获取和分析图像, 以获得 pHi 值。

这里我们描述了在卵泡上皮中测量皮的过程, 这涉及到几个步骤。首先, 卵巢是从适当的基因型果蝇解剖和安装工具 (图 1)。然后利用定量荧光显微镜对 ovarioles 进行成像, 并对图像进行分析, 得到皮的测量结果。对于每个图像, 单元格类型的兴趣被标识为3.1 节 (图 2) 中所述。GFP 和 mCherry 通道中荧光强度的比值, 使用标准曲线 (图 3A) 转换为φ值, 如3.2 节所述。使用这种方法, 我们发现, pHi 增加与分化早期 FSC 谱系, 从6.8 在演, 到7.0 的 prefollicle 细胞, 7.3 在卵泡细胞 (图 3B)。每个单元格类型的φ值可以以图形方式表示, 如图 3B中所示, 或者作为 pseudocolored 显微图像显示 pHluorin 与 mCherry 比率 (图 4和 图 5) 的差异。在这些图像中, pHluorin 与 mCherry 比率的差异显示为颜色的差异, 如查找者所定义的。重要的是要选择一个查找, 最大和最小值范围的颜色, 以产生一个图像, 是最有代表性的数据。虽然一般情况下, 选择 "查找" 和 "范围" 设置不会给出图像中不存在的差异的外观, 但不太适合的尿路可能会掩盖在显微照片中存在的φ差异 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56316/56316fig1.jpg"> 
图 1: 用于解剖和安装的材料果蝇ovarioles.(A) 图像显示: (1) 指甲油;(2) 用于润滑脂的真空润滑脂、烧杯和吸管头;(3) 23 口径注射器针;(4) 不锈钢钳, 大小 5;(5) 3D 印刷安装室;(6) 22 X 40 mM 玻璃片;(7) 圆形玻璃片, 直径12毫米, 厚度 0.13-0. 16 毫米;和 (8) 9-井玻璃解剖盘。(B) 一个3D 打印安装腔的特写图像。(C) 显示解剖对野生卵巢的图像。(D) 显示分离 ovarioles 的图像。(E) 带有解剖卵巢的3D 室的图片, 安装在圆形玻璃片下。黑点是指甲油的滴, 用来举行圆形的片到位。(F) 在安装后的圆形玻璃片下解剖 ovarioles 的图像。黑盒表示图像的一个区域, 有一个单独的解剖 ovariole。刻度线代表大约500µm.<a href="//ecsource.jove.com/files/ftp_upload/56316/56316fig1large.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/56316/56316fig2.jpg"> 
图 2: 识别演、全氟碳化物和卵泡细胞.两个例子的 germaria 与无人使用 mCherry::p hluorin 和 10930-Gal4 染色与 Concanavilin-A。每个图像中都显示了一个 ROI, 它概述了 FSC、pFC 和卵泡细胞。用 pHluorin 和 mCherry 通道的平均荧光强度计算 pHluorin mCherry 比。刻度线代表大约10µm.<a href="//ecsource.jove.com/files/ftp_upload/56316/56316fig2large.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/56316/56316fig3.jpg"> 
图 3: 在 FSC 谱系中分化期间皮的增加.来自 Ulmschneider et al.的代表性结果2显示: (A) 用于计算从 pHluorin 到 mCherry 比值的 pH 值的典型线性回归曲线;和 (B) 计算的φ值, 演、全氟和卵泡细胞的置信区间为95%。此图已在权限之后进行了改编, 从 Ulmschneider et al.2.<a href="//ecsource.jove.com/files/ftp_upload/56316/56316fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56316/56316fig4.jpg"> 
图 4: 使用斐济生成 pseudocolored 比率图像.(A) 显示背景减法过程中每个步骤的结果的图像。从未处理的图像 (左面板) 开始, 第一步是设置阈值限制, 以便排除在背景下有强度值的像素。阈值上限设置为 "最大"。斐济将把被排除的像素变成蓝色, 从而产生带有蓝色背景的图像和清晰可见的 germarium (中间板)。注意, 此步骤是可选的。第二步是在不带信号的图像的一部分 (中间板的红色方块) 中绘制 roi, 测量 roi 的平均荧光强度, 并从整个图像中减去该量, 从而产生背景减去图像 (右面板)。第三步是利用图像计算函数, 将图像在 pHluorin 信道中的图像分割成 mCherry 信道。结果将是一个图像显示与灰色的查阅表格和图像显示值设置为跨越整个动态范围提供的图像的位深度。最后一步是将图像显示设置调整到更合适的范围, 并选择一个查找表。(B) 示例图像, 显示图像计算的结果, 其最小显示值设置为 0, 最大显示值设置为 2.5, 使用四不同的查找表。每个图像旁边的矩形显示了每个查找表的动态范围所使用的颜色。请注意, 对于希洛, 16 色和热, 不同的颜色用于像素的强度值为0或最大 (例如, 蓝色和红色, 分别在希洛)。这为动态范围的限制提供了一个简单的可视化参考, 允许查看器看到信号在动态范围内。(C) 屏幕截图, 显示在使用生物格式导入插件将文件导入 ImageJ 时所选择的选项. 刻度线代表10µm.<a href="//ecsource.jove.com/files/ftp_upload/56316/56316fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
少女 = "jove_content" 佛: 保持在一起. 内页 = "1" ><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56316/56316fig5.jpg"> 
图 5: 设置 pseudocolored 比率图像的图像显示值.应设置最小和最大图像显示值, 以使来自碳酸盐条件的图像中的信号以及两个尼日利亚条件都在动态范围内。为了说明这一点, 使用热查找表显示了三条件中的两个图像, 其中有四不同的图像显示设置 (0-0. 5、0-1、0-2. 5 和 0-5)。请注意, 对于所有的三实验条件, 当图像显示为最大显示值设置为 0.5, 大部分的信号是在或接近最大的色度刻度, 当它被设置为 5.0, 大部分的信号是在或接近最小的颜色imetric 规模。在这两种情况下, 不同的 pHluorin 到 mCherry 比例的组织不能轻易欣赏, 所以这些设置是不理想的。相比之下, 1.0 或2.5 的最大显示值更合适。通过这些设置, 可以很容易地欣赏到组织间比值的差异, 所有三实验条件下的图像中的信号都显示在比色刻度的动态范围内的颜色中。刻度线代表大约10µm.<a href="//ecsource.jove.com/files/ftp_upload/56316/56316fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue at JoVE.com

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

我们为活体果蝇卵巢组织中的上皮干细胞谱系的细胞内 pH 的成像提供了一个协议。我们描述的方法来产生转基因苍蝇表达 ph 生物传感器, mCherry::p hluorin, 图像的生物传感器使用定量荧光成像, 生成标准曲线, 并转换荧光强度值的 ph 值。

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

methods-for-imaging-intracellular-ph-follicle-stem-cell-lineage-live

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

6.8K 次观看.  University of California, San Francisco. 我们为活体果蝇卵巢组织中的上皮干细胞谱系的细胞内 pH 的成像提供了一个协议。我们描述的方法来产生转基因苍蝇表达 ph 生物传感器, mCherry::p hluorin, 图像的生物传感器使用定量荧光成像, 生成标准曲线, 并转换荧光强度值的 ph 值。

视频: Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

成像核内作用棒在活热应力 果蝇 胚胎

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. Here, we describe in detail our current protocol for the dissection of Drosophila larval brains, and then present our current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for high-resolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. We also present custom-made macros that we routinely use to correct for drifting and to extract and process specific quantitative information from time-lapse analysis.

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

This protocol describes applications of sample immobilization using Fibrin clots, limiting drifting, and allowing the addition and washout of reagents during live imaging. Samples are transferred to a drop of Fibrinogen containing culture medium on the surface of a coverslip, after which polymerization is induced by adding thrombin.

使用纤维素凝块固定 的果蝇 组织用于实时成像

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...