The authors would like to thank Dr. Anas Kamleh for valuable help with optimizing mass spectrometry methods, and Annika von Vollenhoven for assistance with cell sorting. This research was supported by the Swedish Foundation for Strategic Research (grant no. FFL12-0220) and the Strategic Programme in Cancer Research (IR, RN); the Swedish Heart-Lung Foundation (CEW, HG); and Mary Kay Foundation (JW, MJ).

1.代謝物の抽出<ol><li> 皿からの抽出 <ol><li>安定同位体標識された培養培地+透析サプリメント（血清または他の成長サプリメント）で三連で6ウェルプレートで培養細胞は、細胞が75％コンフルエントになるまで。 注：U- 13 C-グルコース40％を含むRPMI中で48時間と70％U- 13 C、15 N2-グルタミンおよび5％透析FBS（ウシ胎児血清）のためにここでは、培養HeLa細胞。透析FBSは、標識されたメディアを汚染する可能性がある低分子量代謝物を取り除くために使用されます。実際の実験の前に透析したサプリメントで細胞を培養する培地で細胞を正常に増殖していることを確認することをお勧めします。サプリメントは、ヘビ肌透析チューブを用いて、0.15 M NaCl溶液で一晩透析します。 </li><li>抽出廃棄培地の日に、500μLの冷HBSSで二回、ウェルを洗浄し、それを捨てます。 注：ここでは40％を含むHBSSを使用U-13 C-グルコースは培地もあるからです。 </li><li>追加600μLの100％メタノールをドライアイス上で予備冷却しました。 </li><li>ドライアイスに皿を転送し、セルスクレーパーで細胞材料を除去します。 </li><li>注意深く質量分析まで-80℃でマイクロ遠心チューブとストアに細胞抽出物をピペット。 </li></ol></li><li> モック選別細胞の抽出 <ol><li>安定同位体標識された培養培地+透析サプリメントで三重100 mmディッシュで培養細胞。 注：100ミリメートル皿で48時間ここで、培養HeLa細胞の細胞の数が多い（〜4×10 6）を 50万選別された細胞を得るために必要とされるからです。 HeLa細胞抽出物のこの数は、代謝物の良好な測定を得るために必要でした。培地は、40％U- 13 Cグルコース、70％U- 13 C、15 N 2グルタミン及び5％透析FBSを含むRPMIを用いました。 </li><li>抽出、廃棄培地の日に、Wウェルをすすぎますi番目の1.5mLの温かいHBSSし、それを捨てます。 </li><li> 37℃で4分間、1.5 mLのトリプシン/ EDTAを加えることによって、細胞を切り離します。 4°Cまたは氷で次の手順を実行します。 </li><li> 3 mLの氷冷HBSS（ハンクス液）+透析サプリメントを追加することにより、トリプシンを無効にします。 </li><li> 3分間750×gで15 mLチューブと遠心分離機で細胞を収集します。 </li><li>濃度1〜2×10 6細胞/ mLのHBSS +透析サプリメント+ 1mMのEDTAでペレットを再懸濁し、単一細胞を得るために40μmのセルストレーナーを通過し、5 mLのチューブに移します。 </li><li>ソートのみ一重のためのセルソーター・ゲーティングを介して細胞、および4℃で3分間、750×gで遠心分離選別細胞。 楽器圧力27 psiのと速度千のイベント/秒でここソートHeLa細胞、および100μmのノズルを使用します。注。 HeLa細胞は、それらが可能な限り無傷のペレットを得るために遅い事象率と大ノズルでソートされるべき大です。寒さのブロック内のセルを保つthroughou代謝を減少させるために選別T。手順ソートの完全な説明は、以前12に記載されています。 </li><li>上清を捨て、メタノールを加える前に、均一なペレットを得るために、50μLの氷冷のdH 2 Oにペレットを再懸濁。直接ペレットへの冷メタノールの添加は、再懸濁しにくい固体ペレットを形成しています。 </li><li>ドライアイスに保た540μLのメタノールを添加することにより、代謝産物を抽出し、液体クロマトグラフィーするまで-80℃で抽出を維持 - 高分解能質量分析（LC-HRMS）分析。 </li></ol></li><li> 細胞周期選別された細胞の抽出 <ol><li>培養培地+透析サプリメントで三重100 mmディッシュで培養細胞。 注：GemininのFucciグリーンG1（負）とSG2M（陽性）細胞の選別を可能にします（MAG1-hGem）プローブ13を含む。ここで、培養したHeLa細胞。細胞は用の安定同位体のトレース培地で培養することができます必要に応じて長いですよ。ここでは、我々は40％U- 13 C-グルコースおよび70％U- 13 C、ソート開始する前に、15 N2-グルタミン2時間を含むRPMIに切り替えた、標識されていない培地で46時間のためにそれらを培養しています。これは、短いパルス標識を必要とする細胞周期段階を研究することができるようにするために行きました。 </li><li>抽出、廃棄培地の日に、1.5 mLの温かいHBSSでウェルを洗浄し、それを捨てます。 </li><li> 37℃で4分間、1.5 mLのトリプシン/ EDTAを加えることによって、細胞を切り離します。 4°Cまたは氷で次の手順を実行します。 </li><li> 3 mLの氷冷HBSS +透析サプリメントを追加することにより、トリプシンを無効にします。 </li><li> 3分間750×gで15 mLチューブと遠心分離機で細胞を収集します。 </li><li>濃度1〜2×10 6細胞/ mLのHBSS +透析サプリメント+ 1mMのEDTAでペレットを再懸濁し、単一細胞を得るために40μmのセルストレーナーを通過し、5 mLのチューブに移します。 </li><li>セルソーターグラムをソート細胞その後、破片やダブレットをating関心の細胞マーカーのためのゲーティング、および遠心選別細胞を4℃で3分間、750×gで。 楽器圧力27 psiのと速度千のイベント/秒でここソートHeLa細胞、および100μmのノズルを使用します。注。代謝率を減少させるためにソートを通して冷たいブロック内のセルを保管してください。 </li><li>上清を捨て、メタノールを加える前に、均一なペレットを得るために、50μLの氷冷のdH 2 Oにペレットを再懸濁。直接ペレットへの冷メタノールの添加は、再懸濁しにくい固体ペレットを形成しています。 </li><li>ドライアイスに保た540μLのメタノールを加えて代謝物を抽出し、LC-HRMS分析まで-80℃で抽出物を保持します。 </li></ol></li></ol> 2.質量分析注：ここでは、LC-HRMSシステム上の細胞抽出物を分析するためのプロトコルについて説明します。細胞抽出物の分析のための任意のメタボロミクスの方法を用いることができます。フルスキャン分析は、代謝物の広い範囲を検出するのに有用である可能性があります。 <ol><li>質量分析基準較正ミックスを使用して、機器のキャリブレーションを行います。 </li><li> 15秒間30分間ボルテックス氷上で細胞抽出物を解凍します。 </li><li> 4℃で13,000×gで10分間スピンフィルターや遠心分離機に細胞抽出物の100μLを転送します。 </li><li> LC-HRMSシステムにろ液の12.5μLを注入します。 </li><li> 0.1％のギ酸の勾配溶出を使用して、ZIC-HILICガードカラム（2.1ミリメートル×20ミリメートル）を装着した双性イオン性親水性相互作用液体クロマトグラフィー（ZIC-HILIC）カラム（150ミリメートル×4.6ミリメートル、5μmの粒子サイズ）を使用して、個別の代謝物水中での酸（溶媒A）およびアセトニトリル（溶媒B）。溶剤Aの20％で勾配溶出を開始し、17分で80％まで増加。それぞれ、4 400μL分の流速で分-1および23℃と4℃のカラム温度とサンプルトレイ中にこの割合を維持します。 </li><li>私を使用してください代謝物の検出、イオン化源として正と負の両方のモードで加熱エレクトロスプレー（H-ESI II）、70,000半値幅（FWHM）の質量分解能でフルスキャン取得モードのためのクロマトグラフィー分離に結合nstrument（ M / Z 200）。 </li><li> 45と10のauの流量（任意単位）と設定し、350℃での気化温度とポジティブモードと-3.5 kVの4 kVのでエレクトロスプレー電圧でシースガスおよび補助ガスを窒素（純度&gt; 99.995パーセント）を使用しますネガティブモードインチ</li></ol> 3.データ解析<ol><li>規格が利用可能であり、試料中の良質なピークを示しているために代謝産物の数を選択します。良質のピークは、高い信号対雑音比を有しています。ピークの品質を検証し、偽の同位体を含まないことを確認することが重要です。形状および/または保持時間が異なる同位体ピークはおそらくfalseです。 </li><li>同位体標識されたサンプルについて（質量アイソトポマーを計算MI）すべてのMIの総ピーク面積と各MIのピーク面積を分割することによって画分。 </li><li>それぞれ、13 Cおよび15 Nの濃縮をラベルされた炭素/窒素画分を計算します <img alt="式（1）" src="/files/ftp_upload/55011/55011eq1.jpg" /> ここで、nは、代謝物中の炭素の総数（または窒素、それぞれ）は、ミックスは、xのMI分率です。 注：すべての計算は、プログラミング言語を用いて行うことができます。 </li></ol>

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著者らは、開示することは何もありません。

我々の方法は、細胞代謝産物中のMIDは、細胞の代謝活性の「歴史」を反映している原理に基づいています。これは、細胞の複雑なコミュニティで生じたような従来の細胞選別手順と、細胞の亜集団における代謝活性を調査することができます。これとは対照的に、代謝物のピーク面積は、ソートされた細胞と培養皿11から直接抽出の抽出物との間で著しく異なります。部分?...

高等生物は、より複雑な機能をもたらすために協力異なる細胞型の複雑なコミュニティを含みます。例えば、腫瘍は、癌細胞だけでなく、線維芽細胞、血管を構成する細胞だけでなく、を含み、多くの場合、免疫細胞は、1浸潤します;血液は、免疫細胞サブタイプ2の数十の複雑な混合物が含まれています。さらには培養細胞株は、乳癌細胞3の管腔および基底サブタイプなどの複数の亜集団、から構成されてもよいです。また、共存する異なる細胞型は、代謝、「コラボレーション」を示すことができます。例えば、脳内で、アストロサイトは、この基板4を酸化ニューロンへのその後「摂食」である乳酸にグルコースを変換するために考えられています。 Tリンパ球は、システイン5の供給源として、隣接する樹状細胞に依存して、いくつかの状況です。そして癌細胞はASSOと協力することができます腫瘍6でciated線維芽細胞。このようなシステムの代謝挙動を理解するために、存在する種々の細胞型の代謝活動を分離して測定することが不可欠です。 はるか細胞型を分離するために最も広く使用される方法は、蛍光活性化細胞選別です。この方法は広く適用可能であり、目的の細胞型または状態は、蛍光抗体、操作された蛍光タンパク質の発現、又は他の染料を使用して、「標識された」ことができることを条件とします。 1つのオプションは、次にセルソーター、得られた再培養個々の細胞型、及び貫通最初は別個の細胞タイプは、これらの培養物7の代謝試験を行うことです。細胞型または表現型は、培養条件で安定であり、そのような細胞周期状態を、また、共培養における代謝協力として過渡的な挙動を捕捉することができない場合は、これは実現可能です。このような場合のために、代謝がそうで直接測定されなければなりませんRTED細胞。セルは手順をソートするので、これは困難である彼らの新陳代謝8を歪めることがストレスに細胞を施し、そして我々はこのアプローチ9、10を取るだけ少数の研究を認識しています。特に、我々は絶対的な代謝産物の存在量の測定は、もはや信頼性があるように、アミノ酸等の主要な代謝産物は、細胞ソーティングバッファに保持した細胞から漏れないことを見出した11（ソートされた画分の間の相対的な比較は依然として価値があるかもしれません）。 これらの問題を回避するために、我々は、ソートする前に、安定同位体で細胞を標識し、細胞代謝産物ではなく、代謝物の存在量でのMIDに焦点を当てます。 MIDは長い時間スケール上に形成されているので、それらは以下のソート条件に短期暴露の影響を受けなければなりません。我々は、DAを提供するのに十分な感度でフルスキャンの高分解能質量分析を使用してのMIDを定量化セルソーティング時間の約30〜60分を必要とする50万選別した細胞から始まる代謝物の何百も、上のta。培養皿から直接制御（細胞は、任意の特定の集団をゲーティングすることなく、セルソーター器を通過した）および代謝物の抽出を「モックがソート」との比較は観察のMIDは、元の培養中のものの代表であることを保証するために行われます。安定同位体トレーサーの選択に応じて、様々な代謝経路は、この方法で研究することができます。

<table border="0" cellpadding="0" cellspacing="0" width="1001">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">HBSS</td>
			<td style="width:165px;">Sigma</td>
			<td style="width:139px;">H6648</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INFLUX (inFlux v7 Sorter)</td>
			<td style="width:165px;">BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-13C-Glucose</td>
			<td>Cambridge isotopes</td>
			<td>40762-22-9 / GLC-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-13C,15N2-Glutamine</td>
			<td>Cambridge isotopes</td>
			<td>CNLM-1275-H-0.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol (JT Baker), HPLC grade</td>
			<td style="width:165px;">VWR</td>
			<td style="width:139px;">BAKR8402.2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrafree - MC - VV centrifugal Filters. Durapore PVDF 0.1 &#181;m</td>
			<td style="width:165px;">Millipore</td>
			<td style="width:139px;">UFC30VV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultimate 3,000 UHPLC</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q-Exactive Orbitrap Mass spectrometer</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Merk-Sequant ZIC HILIC column (150 mm x 4.6 mm, 5 &#181;m)</td>
			<td style="width:165px;">Merck KGaA</td>
			<td style="width:139px;">1.50444.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Merk-Sequant ZIC HILIC guard column (20x2.1 mm)</td>
			<td style="width:165px;">Merck KGaA</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile Optima LC-MS, amber glass</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:139px;">A955-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milli-Q water</td>
			<td style="width:165px;">Millipore</td>
			<td style="width:139px;"></td>
			<td>Produced with a Milli-Q Gradient system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MYRSYRA 99.5% OPTIMA (Formic acid)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:139px;">11423423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X100 Screw Vial 1.5ml, 8-425 32x11.6mm,amber, 100 units</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;">10560053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X100 LOCK SKRUV VITT PTFE PACKNING 8-425 (Screw caps)</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;">12458636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProteoMass LTQ/FT-Hybrid ESI Pos. Mode Cal Mix</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">MSCAL5</td>
			<td>Calibration kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SNAKESKIN 10K MWCO&#160;</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;">88245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mathematica v.10&#160;</td>
			<td style="width:165px;">Wolfram Research</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
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a method for measuring metabolism in sorted subpopulations of complex cell communities using stable isotope tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

一例として、ここでは、細胞周期の段階に応じてソートされたHeLa細胞の代謝を調べる実験を記載します。炭素と窒素の両方に中央代謝物の広い範囲を標識するために、我々はU- 13 C-グルコースおよびトレーサーとしてU- 13 C、15 N-グルタミンを使用して48時間、細胞を培養しました。同位体の中間レベルはより多様MIパターン14を</su...

Watch this Scientific Journal Video about A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing at JoVE.com

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

安定同位体のトレースを使用した複雑な細胞のコミュニティのソートされた亜集団で代謝を測定する方法

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

a-method-for-measuring-metabolism-sorted-subpopulations-complex-cell

Research

JoVE Journal

Biochemistry

8.2K ビュー.  Karolinska Institutet. This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

ビデオ: A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

RNAの測定方法<em&gt; N</em<sup&gt; 6</sup細胞や組織における&gt; -methyladenosine変更

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

生化学

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

油中水型エマルションの遠心分離により巨大ベシクルカプセル化微小球の調製

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

酸に不安定な保護基を保持するのに適したNi-シッフ塩基錯体の使用条件の加水分解

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

異種タンパク質複合体-DNA相互作用研究のための改変酵母1ハイブリッドシステム

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

同位体 2 つの安定を用いる液体クロマトグラフィー質量によってスフィンゴミエリンの定量的および定性的な方法ラベル スフィンゴミエリン種

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

植物の鉄含有量を測定するための比色定量法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

マッピング代謝: 監視組織で直接乳酸脱水素酵素活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

安定炭素・酸素同位体分析の炭酸塩のサンプリングと歯のエナメル質の前処理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

測定と酸素消費量全体の解釈飛ぶ頭セグメント

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

CD95死誘導シグナル複合体の組成とプロスパスパーゼ-8の処理の測定

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...