Name: methods for imaging intracellular ph of the follicle stem cell lineage in live drosophila ovarian tissue
Uploaded: 2017-09-26T14:00:00
Description: 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

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 + ...

<ol>
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Cell Biol. 215 (3), 345-355 (2016).</li><li>Han, J., Burgess, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Indicators+for+Intracellular+pH.">Fluorescent Indicators for Intracellular pH.</a> Chemical reviews. 110 (5), 2709-2728 (2010).</li><li>Koivusalo, M., Welch, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amiloride+inhibits+macropinocytosis+by+lowering+submembranous+pH+and+preventing+Rac1+and+Cdc42+signaling.">Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling.</a> J. Cell Biol. 188 (4), 547-563 (2010).</li><li>Choi, C. -. H., Webb, B. A., Chimenti, M. S., Jacobson, M. P., Barber, D. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Kirilly, D., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+ovary:+an+active+stem+cell+community.">The Drosophila ovary: an active stem cell community.</a> Cell research. 17 (1), 15-25 (2007).</li><li>Sahai-Hernandez, P., Castanieto, A., Nystul, T. 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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. 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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>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 (<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 ...

Research

JoVE Journal

Developmental Biology

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

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

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

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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 ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

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

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

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

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

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

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

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

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

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

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

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

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 ...