カイヨーンソン イヴァンコレア アンドレアスブレヒト カトリンMüentener クリスのれん 羅日 明許 セオドアデイビス サルヴァトーレRussello 愛華張 インカゴーシュ エリッサMaunus ニコラスラバルト ジェームズマクファーランド ジョンバスウェル ブレンダデズモンド ジェームズエラード ドンコーム

メッキCHO - K1細胞： <ol><li> F - 12Kの完全の6ミリリットルの標準プロトコールに従ってトリプシン処理されているCHO - K1細胞を50 -100μLを加える。 </li><li>その後、上下に数回ピペッティングして混合細胞懸濁液、＃1.5ホウケイ酸カバーガラスチャンバー（NalgeneヌンクのIntと滅菌8室のLab - Tek社IIチェンカバーガラスの各ウェルにトリプシン処理細胞懸濁液のアリコートを300μl;商品＃155409;ロット：100808-8-0）。 </li><li> 37℃、5％CO 2で一晩試料をインキュベートする。 </li></ol> pSNAP - ADRB2とpSNAP - H2BとCHO - K1細胞のトランジェントトランスフェクション： <ol><li>細胞密度と健康を探すために前日に播種された細胞培養チャンバーを確認してください。 </li><li>マイクロチューブの適切な数のラベルを付けます。 </li><li>層流フードでは、トランスフェクション複合体混合物は、0.3μgのpSNAP - ADBR2コントロールプラスミドDNAまたはpSNAP - H2BのコントロールプラスミドDNA、TransPassのD2を1μlとTransPass V 1μlの0.3μgのいずれかを使用して、次のサンプルチャートに基づいて設定反応あたり。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>プラスミド</td><td>反応の数</td><td>無血清培地の液</td><td> TransPass D2の液</td><td> TransPass Vの添加</td><td>プラスミドDNAの添加</td><td> TransPassのD2/reactionμlの</td><td> TransPass V /反応の液</td><td> DNA /反応のNG </td><td> DNAコンク。 （ng /μlに） </td><td>プラスミドDNA /反応μlの</td></tr><tr><td> SNAP - ADRb2 </td><td> 5 </td><td> 236.5 </td><td> 5 </td><td> 5 </td><td> 3.5 </td><td> 1 </td><td> 1 </td><td> 300 </td><td> 472.34 </td><td> 0.7 </td></tr><tr><td> H2B - SNAP </td><td> 5 </td><td> 237 </td><td> 5 </td><td> 5 </td><td> 3 </td><td> 1 </td><td> 1 </td><td> 300 </td><td> 515.89 </td><td> 0.6 </td></tr><tr><td>モック</td><td> 5 </td><td> 240 </td><td> 5 </td><td> 5 </td><td> 0 </td><td> 1 </td><td> 1 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr><td>トランスフェクトされていない</td><td> 5 </td><td> 250 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr></tbody></table></li><li>最初に、適切なプラスミドDNAを無血清F - 12K混ぜ、その後TransPass Vトランスフェクション試薬を追加し、TransPass D2フェクション試薬を加える。各試薬の添加後、穏やかに数回ピペッティングでよく部品を混ぜる。 </li><li> 30分間室温で反応します。 </li><li>インキュベーションの間、完全DMEM600μlので細胞を1回洗浄し、各サンプルに完全DMEM 450μlを加える。 </li><li>ゆっくりと滴下方式で、各サンプルにトランスフェクション複合体混合物の50μlを追加。 </li><li> ° C、5％CO 2 37℃で一晩サンプルをインキュベートする。 </li></ol>一過性pSNAP - ADRB2とpSNAP - H2BでトランスフェクトしたCHO - K1細胞のSNAP -セルのTMRのスターラベル： <ol><li>慎重に真空吸引することにより、各サンプルからトランスフェクション複合メディアを削除します。 F - 12Kの完全な600μlで細胞を2回洗浄し、F - 12Kの完全な600μlで各ウェルの培地を交換してください。 </li><li>完全なF - 12Kと51μlの0.6 mMのSNAP -セルTMR -スター基板の995μlを添加して色素のラベリングのメディアを混在させる。 SNAP -セルのTMR - Starの最終濃度は、3mmにする必要があります。基質添加後混合して数回上下にピペッティング。 </li><li>慎重にサンプルから増殖培地を除去し、各ウェルに色素ラベリングメディアの200μlを加える。 37サンプルをインキュベート° C、30分間、5％CO 2を 。 </li><li>完全なF - 12Kの10mlに、サンプルはインキュベートされているが、ヘキスト33342、三塩酸塩、三水和​​物（Invitrogen社Molecular Probes社水に10 mg / mlの溶液を、16.2 mM溶液）1μlを混合して試料の核染色用にHoechst 33342を溶液を調製。 </li><li>すべてのサンプルからメディアを取り出して、各サンプルの希釈ヘキスト溶液を600μlずつ加える。 37サンプルをインキュベート° C、5分間、5％CO 2を 。 </li><li> F - 12Kの完全な600μlのサンプルを3回洗浄する。 </li><li> 37サンプルをインキュベート° C、5％非法人の蛍光物質が細胞外に拡散するようにさらに30分間CO 2。 </li><li> 30分後、最後にもう一度は、SNAP -細胞サンプルにメディアを変更し、画像処理に進みます。 </li><li>ツァイス線分Axiovert 200M蛍光顕微鏡を用いて画像の細胞。 </li></ol>代表的な結果<img src="/files/ftp_upload/1876/1876fig1.jpg" alt="図1" /> 図1ここです。SNAPタグ融合を表現しているライブCOS - 7細胞の標準的な蛍光顕微鏡を用いた蛍光イメージングのいくつか代表的な結果。この最初のイメージはSNAP -セルTMR -スターで標識pSNAP - H2Bを（ヒストン核）を発現するライブCOS - 7細胞を示しています。 pSNAP - H2BコンストラクトはpSNAP -タグ（m）のベクトルを用いて生成されました。細胞を30分間SNAP -セルのTMR -スターで標識し、核についてはヘキスト33342（青）で対比染色した。ピンクの蛍光は、ヒストンタンパク質が核を示す青色の蛍光に加えて存在する赤色蛍光のオーバーレイを示しています。 H2Bタンパク質の発現は、明確かつ容易に識別されます。 <img src="/files/ftp_upload/1876/1876fig2.jpg" alt="図2" /> <img src="/files/ftp_upload/1876/1876fig3.jpg" alt="図3" /> 図2と図3。次の2つは画像が一時的にpSNAP -ADRβ2でトランスフェクトしたライブCOS - 7細胞を示す。 細胞は、SNAP -セルTMR -スター（赤）で標識し、ヘキスト33342（青）で対比染色した。 pSNAP -ADRβ2コンストラクトはpSNAP -タグ（m）のベクトルを用いて生成されました。細胞を30分間SNAP -セルのTMR -スターで標識し、核についてはヘキスト33342（青）で対比染色した。青色の蛍光が核を識別しながら、赤色の蛍光は、細胞表面の受容体タンパク質のADRB2の存在を示している。

<ol>
	<li>Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+biochemical+activities+in+living+cells.">Visualizing biochemical activities in living cells.</a> Nat Chem Biol. 5, 63-65 (2009).</li><li>Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAP-tag+Technologies:+Novel+Tools+to+Study+Protein+Function.">SNAP-tag Technologies: Novel Tools to Study Protein Function.</a> NEB Expressions. 3.3, 1-3 (2008).</li></ol>

著者らは、この記事で使用する試薬や器具を生産するニューイングランドバイオラボで採用されています。

フルオロフォアの光安定が退色する前に捕捉されている必要がありますどのように迅速に蛍光を決定するような焦点、レーザー強度、機器の機能、レンズが使用される、などの観測結果に影響を与えるいくつかの変数があります。 タンパク質標識のこの方法は汎用性と、多くのアプリケーションに適しています。合成プローブとの融合タンパク質の特定のラベルのためのSNAP - tagとCLIPタグ技術が生きている細胞で、細胞溶解物の動的なプロセスのさまざまなを含むタンパク質の機能のさまざまな側面を、有効にしてください。 SNAP -、CLIP -、MCP -およびACP -タグ技術は、ライブと固定された細胞中のタンパク質のイメージング、およびin vitroでのタンパク質の研究にシンプルさと異常な汎用性を提供します。単一の遺伝子コンストラクトの作成は、蛍光色素、ビオチン、またはビーズを含む官能基、のさまざまな共有結合を形成することができるタグ付き融合タンパク質が得られます。目的のタンパク質をクローニングし、発現し一度だけして、融合はそのような生細胞内の同時タンパク質標識、タンパク質の局在や転座、パルス - チェイス実験、受容体の内在化の研究など、数多くのダウンストリームアプリケーションのための蛍光基質の様々で使用することができますする必要があります。 、選択的な細胞表面のラベリング、タンパク質はアッセイ、SDS - PAGEにおけるタンパク質の検出、フローサイトメトリー、高スループットのマイクロタイタープレートに結合アッセイ、バイオセンサーの相互作用の実験、およびFRETに基づく結合アッセイをプルダウン。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CHO-K1 Cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CCL-61</td>
<td>&nbsp;</td>
<tr>
<td>Serum free F-12K</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lab-Tek II Chambered Coverglass with #1.5 borosilicate coverglass chambers</td>
<td>&nbsp;</td>
<td>Nalgene Nunc Int.</td>
<td>155409</td>
<td>Lot: 100808-8-0</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342, trihydrochloride, trihydrate (10 mg/ml solution in water, 16.2 mM solution)</td>
<td>&nbsp;</td>
<td>Invitrogen Molecular Probes</td>
<td>H3570</td>
<td>470519</td>
</tr>
<tr>
<td>Zeiss Apotome Axiovert 200M fluorescent microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Transpass V</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M2561S</td>
<td>0030709</td>
</tr>
<tr>
<td>Transpass D2</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M2554S</td>
<td>0060802</td>
</tr>
<tr>
<td>SNAP-Cell TMR Star</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>S9105S</td>
<td>0010811</td>
</tr>
<tr>
<td>pSNAP-ADBR2 Control Plasmid</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N9178S</td>
<td>0010811</td>
</tr>
<tr>
<td>pSNAP-H2B Control Plasmid </td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N9179S</td>
<td>0010811</td>
</tr>		</tbody>
		</table>
		</div>

fluorescent labeling of cos-7 expressing snap-tag fusion proteins for live cell imaging

SNAP - tagとCLIP -タグタンパク質のラベリングシステムは生細胞で目的のタンパク質に、蛍光色素を含む分子の特定の、共有結合を、有効にしてください。これらのシステムは、イメージング計測の範囲に最適化された蛍光基質の幅広い選択を提供する。一度クローン化と発現し、タグ付けされたタンパク質は、再度クローンを作成することなく、多くのダウンストリームアプリケーションのための基板の様々な使用することができます。このシステムを使用する2つの手順があります。SNAPタグ融合、及び選択肢のSNAP - tagを基板との融合のラベルとして、目的のタンパク質のクローニングおよび発現。 SNAP - tagは、人間のOに基づいて小さなタンパク質である<sup&gt; 6</supDNA修復タンパク質、（hAGT）&gt; - alkylguanine - DNA - alkyltransferase。 SNAPタグラベルは、ベンジルリンカーを介してグアニンに結合した染料またはピリミジン脱離基である。ラベリング反応で、基質の置換ベンジル基が共有結合SNAPタグに添付されます。 CLIP - tagはbenzylcytosineではなく、ベンジルグアニン誘導体と反応するように設計さSNAP - tagの修正版です。 SNAPタグと組み合わせて使用​​すると、CLIP - tagは、同じセル内で同時に2つのタンパク質の直交すると補完的なラベリングが可能になります。

Watch this Scientific Journal Video about Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging at JoVE.com

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP - tagとCLIP -タグタンパク質のラベリングシステムは生細胞で目的のタンパク質に、蛍光色素を含む分子の特定の、共有結合を、有効にしてください。一度クローン化と発現し、タグ付けされたタンパク質は、再度クローンを作成することなく、多くのダウンストリームアプリケーションのための基板の様々な使用することができます。

COS - 7の蛍光標識は、生細胞イメージングのためにSNAP - Tag融合タンパク質を発現する

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

fluorescent-labeling-cos-7-expressing-snap-tag-fusion-proteins-for

Research

JoVE Journal

Biology

22.9K ビュー.  New England Biolabs. SNAP - tagとCLIP -タグタンパク質のラベリングシステムは生細胞で目的のタンパク質に、蛍光色素を含む分子の特定の、共有結合を、有効にしてください。一度クローン化と発現し、タグ付けされたタンパク質は、再度クローンを作成することなく、多くのダウンストリームアプリケーションのための基板の様々な使用することができます。

ビデオ: Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

生物学

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

蛍光を経由してF -アクチンダイナミクスの生細胞イメージングでは、顕微鏡のスペックル（FSM）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

の蛍光標識ショウジョウバエハートの構造

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

キメラゼブラフィッシュ胚における網膜と脳の開発のライブイメージングのためのGFP発現割球の移植

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

生細胞で蛍光タンパク質とのデュアルプローブ光の強調表示を精製の光変換

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA損傷応答タンパク質の2次元および3次元生細胞イメージング

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

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

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

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

でGFP標識タンパク質のイメージングを生きる<em&gt;ショウジョウバエ</em&gt;卵母細胞

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

蛍光タンパク質を発現する生きたマウスでのイメージング細胞内の構造物の生体顕微鏡

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

マウス骨髄由来の樹状細胞とマクロファージの蛍光融合タンパク質の発現

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...