MFR. AJR を DK100227 は、T32 DK007013 によって支えられ、この仕事は、NIH の DK092408 によって支えられました。著者は、CapaR GAL4 と c724 GAL4 博士ジュリアン A.T. ダウを感謝したいショウジョウバエ株。我々 はまた実験飛ぶ十字架を維持支援のためヤコブ B のアンダーソン氏を感謝します。

著者が明らかに何もありません。

ショウジョウバエMTs で pH は私の定量化の成功は健康抽出された MTs と取り付けと郭清 (図 A - C) の質によってまったく異なります。したがって、組織としての慎重な取り扱いは説明が不可欠です。新鮮な PLL でコーティング スライド大幅にソリューションにさらされて以前のスライド援助 MT マウントよりもはるかに多くの接着剤をする傾...

このプロトコルの目的は、遺伝子にコードされた pH インジケーター (GEpHI) を用いた細胞内 pH (pHi) の定量化と基底モデル昆虫 (+ H+輸送を評価するためのこのメソッドの使用方法を示すキイロショウジョウバエ) 腎構造のマルピギー氏管 (MT)。MTs は、ショウジョウバエの排泄器官として、いくつかのキーの点1に哺乳類のネフロンに機能的に似ています。MTs は、胸郭、ハエの腹部の (前部と後部) の尿細管の 2 組として配置されます。各 MT の単一細胞上皮管が根尖を明確なの代謝活性の主細胞から成る (内腔) 介在の星状細胞と同様、基底 (hemocoel) 極性と。発達個別のセグメント、特に初期拡張尿管2に結合分泌の主要なセグメント、経過セグメントは、セグメントおよび形態学的、機能的には、3 前方 MTs が構成されます。携帯電話のスケールで内腔の上皮細胞のイオン輸送、頂端膜 V-atpase3アルカリ金属/H+交換と基底 Na+K+で実現-atp アーゼの4、チャンネル5、Na+は、内向き整流性 K+ -Cl−/HCO3−交換 (NDAE1)6、および Na+K+を駆動-2 Cl−共輸送 (NKCC;Ncc69)7、星状細胞仲介 Cl- 、水輸送8,9。この複雑なアクセス可能な生理学的システムは、ショウジョウバエの遺伝学的および行動の多様なツールセットと組み合わせた場合の内因性イオン伝導機構の調査のための優秀な機会を提供します。
このプロトコルのための理論的根拠は動作し、他のモデルのシステム ツールのエクスポートの統合セルからの潜在性の上皮膜イオン輸送を研究するため遺伝子組み換え可鍛性システムを記述します。PHerry10の式、グリーンの pH に敏感な超黄道 pHluorin11,12 (セフ) と赤の pH に依存しない mCherry13MTs での融合から派生した GEpHI を許可の H+輸送の定量化高 K+/nigericin 校正テクニック14を介して単一の mt 野細胞。多くのイオン輸送体は、H+同等を移動、細胞内 pH は私の定量化はさまざまなトランスポーターを介してイオン移動の関数表現として機能します。ショウジョウバエMT モデル システムはまた組織特異遺伝子15と細胞イメージング全体オルガン試金17と組み合わせて使用することができますが、RNA 干渉 (RNAi)16式の強力な遺伝的ツールを提供しています,18,分子から行動への垂直統合の堅牢なツールセットを作成する尿細管機能の19 。これは対照的に他の多くのプロトコルに歴史的にそのような測定が複雑に依存している、上皮の生物学の評価とマイクロ郭清、洗練されたイオン選択性電極20,21, を大変なの略します。高価な pH 感受性色素を22制限荷重要件と異種組織の細胞特異性悪い。GEpHIs は、広く pH私携帯型23の様々 な測定に使用されています。初期の作品は固有 pH 感受性の緑色蛍光タンパク質 (GFP) pH私培養上皮細胞24を監視するを悪用されるが、過去 20 年を見た使用ニューロンで25、グリア26、菌類27 GEpHIs、植物細胞28。GAL4/UA 式システム15とショウジョウバエMT の生理ユーザー補助の遺伝的構造の細胞ターゲットの可能性の組み合わせはこの pHの調査のための理想的な準備をする私規制と上皮膜イオン輸送。
pH私規制は何十年も研究されている、生活に不可欠です。MT の準備では、pH私の調節の生理学を教えるが、また実行する堅牢なモデル洗練された pHi規制前のヴィヴォとin vivoの調査を提供しています。このプロトコルはショウジョウバエNH4Cl パルス酸法21の読み込みを使用して MT の上皮細胞の基底膜で H+運動の定量化をについて説明しますが、pH 指示薬としては遺伝子エンコードすると、任意の影響を受けやすい遺伝子導入とライブ イメージングの準備にこれらのメソッドとその理論的枠組みを適用できます。

<table>
	<tbody>
		<tr>
			<td>Poly-L-Lysine Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4832</td>
			<td>Store at 4 &#176;C, can be reused.</td>
		</tr>
		<tr>
			<td>Nigericin Sodium Salt</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>CAUTION: Handle with gloves. Store as aliquots of 20 mM stock solution in DMSO at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Adhesive Perfusion Chamber Covers, adhesive size 1 mm, chamber diameter &#215; thickness 9 mm &#215; 0.9 mm, ports diameter 1.5 mm</td>
			<td>Sigma-Aldrich</td>
			<td>GBL622105</td>
			<td>Can be substituted as needed to match perfusion system.</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Ellsworth Adhesives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Helping Hands Soldering Stands</td>
			<td>Harbor Freight Tools</td>
			<td>60501</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Open Gravity-fed Perfusion System with Valve Controller, 8 to 1 Manifold and Reserviors</td>
			<td>Bioscience Tools</td>
			<td>PS-8S</td>
			<td>Any comparable perfusion system can be used.</td>
		</tr>
		<tr>
			<td>Flow Regulator</td>
			<td>Warner Instruments</td>
			<td>64-0221</td>
			<td>Can be substituted as needed to match perfusion system.</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>21720024</td>
			<td>Store at 4 &#176;C in sterile aliquots.</td>
		</tr>
		<tr>
			<td>#5 Inox Steel Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Can be substituted based on experimenter comfort.</td>
		</tr>
		<tr>
			<td>35 mm x 10 mm polystyrene Petri dish</td>
			<td>Corning Life Sciences</td>
			<td>Fisher Scientific 08-757-100A</td>
			<td>Exact brand and size are unimportant.</td>
		</tr>
		<tr>
			<td>75 x 25 mm Microscope Slides</td>
			<td>Corning Life Sciences</td>
			<td>2949-75X25</td>
			<td>Exact brand and size can vary as long as perfusion wells are compatible.</td>
		</tr>
		<tr>
			<td>Filimented Borosilicate Capillary Glass, ID 1.5 mm, OD 0.86 mm, thickness 0.32 mm</td>
			<td>Warner Instruments</td>
			<td>64-0796</td>
			<td>Filiment not necessary, glass can be substituted to match perfusion tubing and perfusion wells.</td>
		</tr>
		<tr>
			<td>Tygon Tubing, ID 1/16 inch, OD 1/8 inch, thickness 1/32 inch</td>
			<td>Fisher Scientific</td>
			<td>14-171-129</td>
			<td>Available from multiple vendors, can be substituted to match perfusion system.</td>
		</tr>
		<tr>
			<td>Vacuum Silicone Grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Plastic Flow Control Clamp</td>
			<td>Fisher Scientific</td>
			<td>05-869</td>
			<td>Available from multiple vendors, sterility not required</td>
		</tr>
		<tr>
			<td>Glass rods, 5 mm diameter</td>
			<td>delphiglass.com</td>
			<td>9198</td>
			<td>Exact size is personal preference, multiple vendors available</td>
		</tr>
		<tr>
			<td>PAP Hydrophobic Pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Sealing Film</td>
			<td>Sigma-Aldrich</td>
			<td>P7668</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>HEPES; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>MES; 4-Morpholineethanesulfonic acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>69892</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>TAPS; N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T5130</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>10x/0.45 Air Objective</td>
			<td>Zeiss</td>
			<td>000000-1063-139</td>
			<td>Comparable objectives can be substituted. 40x objectives can be used for single cell imaging.</td>
		</tr>
		<tr>
			<td>Dissecting Stereoscope</td>
			<td>Zeiss</td>
			<td>Discovery.V8</td>
			<td>Any dissecting stereoscope can be used.</td>
		</tr>
		<tr>
			<td>UAS-pHerry transgenic Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 10</td>
		</tr>
		<tr>
			<td>capaR-GAL4 driver line Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 32</td>
		</tr>
		<tr>
			<td>c724-GAL4 driver line Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 2</td>
		</tr>
		<tr>
			<td>Monochromatic High Sensitivity Digital Camera</td>
			<td>Zeiss</td>
			<td>Axiocam 506 mono</td>
			<td>Exact brand and model can vary, can be replaced with any monochromatic high-sensitivity camera suited to live cellular imaging.</td>
		</tr>
		<tr>
			<td>GFP/FITC filter set, 470/40 nm ex., 515 nm longpass em., 500 nm dichroic</td>
			<td>Chroma</td>
			<td>CZ909</td>
			<td>Any GFP/FITC filer set can be substituted.</td>
		</tr>
		<tr>
			<td>RFP/TRITC filter set, 546/10 nm ex., 590 nm longpass em., 565 nm dichroic</td>
			<td>Chroma</td>
			<td>CZ915</td>
			<td>Any GFP/FITC filer set can be substituted.</td>
		</tr>
		<tr>
			<td>Inverted Epifluoescent Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z.1</td>
			<td>Any comparable microscope with motorized filter switching can be used. Upright microscopes can be used with open perfusion baths and water-immersion objectives.</td>
		</tr>
		<tr>
			<td>Statistical Analysis Software</td>
			<td>Microcal</td>
			<td>Origin 6.0</td>
			<td>Any software with comparable functionality can be substituted</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>National Institutes of Health</td>
			<td>ImageJ 1.50i</td>
			<td>Any software with comparable functionality can be substituted</td>
		</tr>
		<tr>
			<td>Image Acquisition Software</td>
			<td>Zeiss</td>
			<td>Zen 1.1.2.0</td>
			<td>Any software with comparable functionality can be substituted</td>
		</tr>
		<tr>
			<td>Single-edged Carbon Steel Razor Blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>71960</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Microscopy Slide Folder</td>
			<td>Fisher Scientific</td>
			<td>16-04</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Fisher Scientific</td>
			<td>50-110-1231</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Polystrene Drosophila Rearing Vials with Flugs</td>
			<td>Genesee Scientific</td>
			<td>32-109BF</td>
			<td>Comparable items can be substituted.</td>
		</tr>
		<tr>
			<td>2.5 L Laboratory Ice Bucket</td>
			<td>Fisher Scientific</td>
			<td>07-210-129</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>NMDG; N-Methyl-D-glucamine</td>
			<td>Sigma-Aldrich</td>
			<td>M2004</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>200 uL barrier pipette tips</td>
			<td>MidSci</td>
			<td>AV200</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>200 uL variable volume pipette</td>
			<td>Gilson Incorporated</td>
			<td>PIPETMAN P200</td>
			<td>Available from multiple vendors.</td>
		</tr>
	</tbody>
</table>

optical quantification of intracellular ph in drosophila melanogaster malpighian tubule epithelia with a fluorescent genetically-encoded ph indicator

上皮膜イオン輸送は本質的な細胞の電気化学勾配の維持と同様、全身のイオンの恒常性に不可欠です。多くのイオン輸送体 (pH私) 細胞内 pH の影響を受けて、トランスポーター活性を評価するための便利なツールは、このように pH は私を監視します。モダンな遺伝子にエンコードされた pH-指標 (GEpHIs) は細胞および細胞内の規模で pH私そのままなセルの光学的定量化を提供します。このプロトコルは細胞 pH私の規制のキイロショウジョウバエのマルピギー氏管 (MTs) で前のヴィヴォライブ イメージングによる pHerry、pKで擬似レシオ メトリック GEpHI リアルタイム定量化をについて説明します、細胞質の pH 変化を追跡に適しています。抽出された大人のフライ MTs は単一細胞層上皮の形態学的、機能的に独立したセクションで構成され、上皮の輸送の調査のためのアクセス可能で、遺伝的に扱いやすいモデルとして利用できます。GEpHIs は、従来の pH に敏感な蛍光染料とイオン選択性電極上のいくつかの利点を提供しています。GEpHIs 適切な促進者の要素が使用異なる細胞集団にラベルを付けることができます。この方、前のヴィヴォ体内、および本質的に不均一であるその場で準備に便利です。GEpHIs もそのままティッシュで繰り返される色素の治療や組織の外部化を必要とせず時間をかけて pHiの定量化を許可します。現在の GEpHIs の主な欠点は、組織の損傷の応答のゾル性細胞質包有物の集約し、過剰発現を構築する傾向です。抽出されたフライ MTs の機能的に異なるプリンシパルと星状細胞に基底プロトン (H+) 輸送の評価を通じて、この議定書では、これらの欠点、彼らのソリューション、および GEpHIs の固有の利点を示した。技術と説明する分析が容易に適応できる幅広い脊椎動物と無脊椎動物の準備と特定のトランスポーターを介してイオン流束の複雑な測定の演習を教えるからアッセイの洗練されたことができます。

健康な組織や前方の MTs を適切に識別、このプロトコルの成功に不可欠です。解剖時に注意が必要なく、直接タッチする MTs とだけハンドルに直接 MTs をグリップとして尿管によってそれらは (図 4 a- B) の破損に 。MTs はスライドにフラットを掃引、細管を可能な限り少し触れられなければならないし、これを回避余分な?...

Watch this Scientific Journal Video about Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator at JoVE.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

細胞のイオン輸送は細胞内 pH モニタリングによってしばしば評価される(pH私)。遺伝子 Encoded pH 指示薬(GEpHIs)は、そのまま細胞内 pH の光学的定量化を提供します。このプロトコルは細胞前のヴィヴォライブ イメージング pHerry とキイロショウジョウバエのマルピギー氏管の細胞内 pH の定量化の詳細については、擬似レシオ メトリック遺伝子にエンコードされた pH インジケーター。

蛍光遺伝子にコードされた pH インジケーターとキイロショウジョウバエのマルピギー氏管の上皮細胞で光の定量化の細胞内 pH

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

optical-quantification-intracellular-ph-drosophila-melanogaster

Research

JoVE Journal

Biology

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

10.1K ビュー.  Mayo Clinic College of Medicine. 細胞のイオン輸送は細胞内 pH モニタリングによってしばしば評価される(pH私)。遺伝子 Encoded pH 指示薬(GEpHIs)は、そのまま細胞内 pH の光学的定量化を提供します。このプロトコルは細胞前のヴィヴォライブ イメージング pHerry とキイロショウジョウバエ...

ビデオ: Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

生物学

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

における寿命の測定<em&gt;キイロショウジョウバエ</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

液胞と細胞質のpHの測定<emインビボで&gt;</em&gt;酵母細胞懸濁液に

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

遺伝的にコードされた分子プローブは、Gタンパク質共役受容体を研究する

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

で液分泌とイオンフラックス速度を測定するためのラムゼイアッセイの使用<em&gt;キイロショウジョウバエ</em&gt;マルピーギ管

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

のメンテナンス<em&gt;キイロショウジョウバエ</em&gt;人口ケージ

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遺伝的にコードされた蛍光一酸化窒素の応用（•NO）シングル細胞におけるNO•信号のリアルタイムイメージングのためのプローブ、geNOps、

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

キイロショウジョウバエ成虫の間接飛翔筋の光学断面筋決定

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...