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?...

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>&lt;strong&gt;Fly Stocks&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals for Buffer preparation&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Dissection and mounting tools&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other equipment&lt;/strong&gt;</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&amp;times; 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 (<stro...

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

Universidad de Cartagena UDEC

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-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

使用果蝇S2细胞进行细胞分裂的活成像

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

遗传学

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

使用实时成像可视化氧化损伤对果蝇卵室的影响

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

发育生物学

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

果蝇卵母细胞的核迁移

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

生物学

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

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

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