当社は、単語 &quot;アナスタシス」アポトーシスの逆転を記述するための示唆のために牧師先生ラルフBohlmannと牧師先生ジェームスVoelzに感謝、安定してシトクロム c -GFPを表現するHeLa細胞についてダグラス·R·グリーン; H446細胞用のチャールズM.ルーディンとエリックE.ガードナー。ビデオでの漫画の図面をアシストするためにヘザーラム。この原稿の貴重な議論のためのイーホイヨ。この作品はサー·エドワード·ユード記念フェローシップ（HLT）、博士ウォルターSzeto記念奨学（HLT）、フルブライト助成金007から2009（HLT）、ライフサイエンス研究財団フェローシップ（HLT）によってサポートされていました、NIHはNS037402（JMH）付与とNS083373（JMH）、および香港大学補助金委員会のAoE / B-07/99（MCF）。ホーラム唐は、生命科学研究財団のShurlとケイクルチ財団フェローである。

The authors have nothing to disclose.

アナスタシスは、細胞死経路を活性化した細胞がその後死んでプロセスを逆にして生き残る現象を指す。ここでは、ライブセルイメージングが実際に同一の個々の細胞は後期アポトーシス細胞死のプロセスを逆にし、次いで生存及び再生し続けることができることを確認するために使用され得ることを実証した。我々のプロトコルは、アポトーシスを誘導し、細胞の大部分は、検証のために?...

アポトーシス（「死に立ち&quot;のギリシャ語）は、一般的に細胞自殺1-7で終わる一方向のプロセスであると仮定される。すでにアポトーシス経路8,9を開始した細胞を含むそれ以外の動物全体で死んでしまう余分な細胞の生存率のプロ死遺伝子結果の遺伝子破壊。同様に、遺伝子操作は、人工的に表示される信号を「私を食べる」、またはそれは、それらの細胞外マトリックスへの接着性を失い、健康、哺乳動物細胞は、細胞全体の食作用またはentosis、それぞれ10,11による死を免れることができます。しかし、その他の遺伝子操作することなく、正常な健康な哺乳動物細胞および細胞株は、アポトーシス12-15の初期段階から回復することができることを示している。細胞はその典型的な重要なチェックポイントに合格した後に個々の細胞を追跡するためのツールを使用して、我々はさらに、アポトーシス12,13の後期段階からの回復を実証しているLY 2-6「ノーリターンのポイント」をマークします。後期アポトーシスのこれらのチェックポイントは、ミトコンドリアのシトクロムc の放出、カスパーゼの活性化、核の断片化、およびアポトーシス体の形成が含まれる。我々は、細胞死2-6の瀬戸際でアポトーシスのこの逆転を記述するために、「生活に上昇」を意味し、ギリシャ語の複合語「アナスタシス」を採用した。 全体瀕死回復プロセスは、ライブセルイメージングによって観察されていない限り、それはアポトーシス事象を経験したことのない細胞からアナスタシスを受けた細胞を区別するために挑戦している。仕事の数十年は、アポトーシスによる細胞自殺の形態学的特徴は進化的に保存され、生化学的および分子事象16〜19によって駆動されることを明らかにした。これらのイベントは、損傷またはdを排除することにより、単細胞と多細胞生物の発達とhomoeostaticプロセスを調節するために細胞の自己破壊を促進するangerous細胞16-19。アポトーシス細胞は、容易に標準化された形態学的に、アポトーシス1,5,6,16,20の生化学的および分子症状によって区別することができるが、現在アナスタシス12,13に固有の既知のマーカーは存在しない。重要なことは、アナスタシスを受けた細胞は、正常な健康な細胞、およびだけアポトーシスを逆転開始細胞として表示され、アポトーシス死細胞12,13のように見える。このように、新しいツールが与えられた生存細胞は、以前にアクティブなアポトーシス過程を経験したことを確実に締結することが必要である。 それが迅速かつ大規模な破壊プロセスであるため、アポトーシスは、一般的に不可逆的なカスケードとして想定されます。ミトコンドリアは、細胞質ゾル21,22へのシトクロム cなどのアポトーシス誘発要因をリリースした後、いくつかの細胞は、アポトーシスを開始することが日に数分かかることがありますが、カスパーゼは、5分23,24内で活性化細胞質が続くことができるとその後すぐに10分25〜27、及び細胞死内の核凝縮25-27。活性化カスパーゼはそのようなエンドヌクレアーゼ阻害剤DFF45 / ICAD 29,30などの細胞解体2,28、の目的のために重要な構造と機能部品を切断し、不活性化することによりアポトーシスを調整する。カスパーゼはまた、 シトクロム c 31,32のミトコンドリアの放出を促進するために、ミトコンドリアに移行し、BCL-2ファミリーメンバーBID、などのプロアポトーシス因子を活性化する。カスパーゼ活性はまた、貪食33を介してマクロファージまたは隣接セルによる死細胞の貪食を促進するための「私を食べる」信号としてホスファチジルセリンの細胞表面露出をもたらす。さらに、アポトーシス事象は、細胞の生体エネルギーと代謝34,35,36を破壊し 、ミトコンドリアが機能不全にレンダリング。したがって、このような破壊からの回復は、直感的には考えにくい。 オリジナルとは逆に期待管理ポイント、細胞がさえ後期アポトーシス細胞死の過程を逆にすることができます。連続的に培養物中の死細胞の運命をモニターすることにより、我々は、初代細胞および細胞株12,13の範囲内の後期アポトーシスの可逆性を観察した。死刺激の除去は、このようなミトコンドリアの断片化、クロマチン凝縮、DNA損傷、原形質膜小疱形成、ホスファチジルセリンの細胞表面露出、ミトコンドリアのチトクロームc の放出、カスパーゼ活性化、核の断片化、細胞収縮のようなアポトーシスの明白な特徴からの回復を可能にしたとアポトーシス体の形成。これらの観​​察は、アナスタシスの機能、結果、およびメカニズムに関する未回答の質問を上げる。これらの質問に対処するために、前提条件は、確実にアナスタシスを受けた細胞を同定することである。ここでは、以前に後期アポトーシスおよび第反転した細胞を検出するためのライブ顕微鏡法およびカスパーゼバイオセンサーを記載ENが生き残った。

<table border="0" cellpadding="0" cellspacing="0" width="667">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Name of Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">LSM780 confocal microscopy</td>
			<td style="width:139px;">Carl Zeiss</td>
			<td style="width:127px;">/</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Glass bottom culture dish</td>
			<td style="width:139px;">MatTek Corporation</td>
			<td style="width:127px;">P35G-0-14-C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Transparent CultFoi</td>
			<td style="width:139px;">Carl Zeiss</td>
			<td style="width:127px;">000000-1116-084</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">CO2 independent medium</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">18045-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">CellTracker</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">C34552</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mitotracker Red CMXRos</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">M-7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hoechst 33342</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">H1399</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Fluorescently labeled annexin V</td>
			<td style="width:139px;">Biovision</td>
			<td style="width:127px;">K201</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis

アナスタシス（「生活に上昇する」ためのギリシャ語）は、死細胞の回復を指します。これらの細胞が回復する前に、それらはミトコンドリアの断片化、細胞質ゾルへのミトコンドリアのチトクロームc の放出、カスパーゼの活性化、クロマチン凝縮、DNA損傷、核の断片化、原形質膜小疱形成、細胞収縮、細胞表面の曝露を含む、アポトーシスの重要なチェックポイントを通過したホスファチジルセリン、およびアポトーシス体の形成。アポトーシス刺激、それによって瀕死細胞がアポトーシスと潜在的に他の死のメカニズムを逆にすることができ、死の前に削除されたときアナスタシスが発生する可能性があります。したがって、アナスタシスも不適切に損傷した細胞を維持することができ、生理的治癒過程を含むようである。アナスタシスの機能とメカニズムは明らかに健康な細胞を回収した後、過去のイベントを検出するための限られたツールによって部分的に妨げられて、まだ不明である。 anastaを検出するための戦略SISはアナスタシスを変調するために生理学的メカニズムの研究、疾患の病状でアンデッドの細胞の危険性、および潜在的な治療が可能になります。ここでは、生細胞顕微鏡および哺乳動物細胞でアナスタシスを特定し、追跡するための哺乳類のカスパーゼバイオセンサーを使用して効果的な戦略について説明します。

アポトーシスの反転を研究するために、組織培養細胞は、最初にアポトーシスを誘発する死刺激にさらされている。細胞がアポトーシスの特徴を表示するとき、新鮮な培養培地は、次いで、刺激を洗い流し、その後回復した （ 図1A）を可能にするために死細胞を培養するために適用される。ここでは、アドレス指定されている重要な問題は、個々の死にかけ培養細?...

Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

トラッキングアナスタシス、アポトーシスを逆に細胞生存現象のための戦略

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

strategies-for-tracking-anastasis-cell-survival-phenomenon-that

Research

JoVE Journal

Biology

17.9K ビュー.  Johns Hopkins University Bloomberg School of Public Health. The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis. 

ビデオ: Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

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, ...

生物学

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

細胞死の正確な評価のために修飾されたアネキシンV /ヨウ化プロピジウムのアポトーシスアッセイ

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

初期のニワトリ胚の形態形成の組織の変形を追跡

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

細菌の凍結乾燥と細胞の生存への影響のための処方

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

モザイク大人のライブイメージングのためのトマト/ GFP-FLP / FRT方法<em&gt;ショウジョウバエ</em&gt;視細胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

トラブルシューティングの方法と現代の定量的蛍光ウェスタンブロッティングへのガイド

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...