카이 Johnsson 이반 코레아 안드레아스 브레히트 카트린 Müentener 크리스 노렌 루오 일 밍 쑤 테오도르 데이비스 살바토레 Russello Aihua 장 잉카 가쉬 엘리사 Maunus 니콜라스 라바흐뜨 제임스 맥팔랜드 존 버스웰 브렌다 데즈먼드 제임스 엘라드 돈 빗

CHO - K1 세포를 도금 : <ol><li> F - 12K 전체의 6ml 표준 프로토콜에 따라 trypsinized되었습니다 CHO - K1 세포의 50 100μl 추가합니다. </li><li> 여러 번 아래 pipetting과에 의해 세포 현탁액을 혼합, # 1.5 borosilicate coverglass 실 (과 살균 8 실 랩 - 테크 II Chambered Coverglass 각 잘하는 trypsinized 세포 현탁액의 다음 나누어지는 300 μl Nalgene Nunc의 INT;. 제품 # 155409; 롯 : 100808-8-0). </li><li> 37 ° C, 5 % CO 2 밤새에서 샘플을 품어. </li></ol> pSNAP - ADRB2 및 pSNAP - H2B와 CHO - K1 세포의 과도 Transfection : <ol><li> 세포 밀도와 건강을 찾기 위해 전날에 씨앗을 품고 세포 배양 챔버를 확인합니다. </li><li> microfuge 튜브의 해당 번호를 라벨. </li><li> 층류 후드에서 transfection 복잡한 혼합물은 0.3 μg pSNAP - ADBR2 제어 플라스미드 DNA 또는 pSNAP - H2B 제어 플라스미드 DNA, TransPass의 D2 1 μl와 TransPass V 1 μl의 0.3 μg 중 하나를 사용하여 다음 예제 차트에 따라 설정 반응 당. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 플라스미드 </td><td> 반응의 수를 </td><td> 혈청이없는 매체의 μl </td><td> TransPass의 D2의 μl </td><td> TransPass V의 μl </td><td> 플라스미드 DNA의 μl </td><td> TransPass의 D2/reaction의 μl </td><td> TransPass V / μl 반응 </td><td> DNA / 반응 NG </td><td> DNA CONC. (μl / NG) </td><td> 플라스미드 DNA / μl 반응 </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> 조롱하다 </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> Untransfected </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> 첫째, 적절한 플라스미드 DNA와 F - 12K 혈청 무료 후 TransPass V의 Transfection 시약을 추가 후, TransPass D2 Transfection 시약을 추가 섞는다. 각 시약의 추가 후 부드럽게 여러 번하고 아래로 pipetting으로 잘 구성 요소를 섞는다. </li><li> 30 분 실온에서 반응을 품어. </li><li> 부화하는 동안, 전체 DMEM 600 μl로 한 세포를 씻어 각 샘플을 완료 DMEM의 450 μl를 추가합니다. </li><li> 천천히, 그리고 드롭 현명한 방식으로, 각 샘플에 transfection 복잡한 혼합물의 50 μl를 추가합니다. </li><li> ° C 5 % CO 2 37 밤새 샘플을 품어. </li></ol> Transiently pSNAP - ADRB2 및 pSNAP - H2B와 Transfected CHO - K1 세포의 SNAP - 셀 TMR 스타 라벨링 : <ol><li> 조심스럽게 진공 흡입하여 각 샘플에서 transfection 복잡한 미디어를 제거합니다. F - 12K 전체 600 μl로 두 번 세포를 씻고 F - 12K 전체 600 μl와 함께 각 잘 미디어를 교체하십시오. </li><li> 전체 F - 12K 51 μl 0.6 MM SNAP 세포 TMR - 스타 기판의 995 μl를 추가하여 염료 라벨 미디어를 섞는다. SNAP - 셀 TMR - 스타의 최종 농도는 3mM해야합니다. 기판의 추가 후 혼합을 여러 번 위아래로 피펫. </li><li> 조심스럽게 샘플에서 성장 미디어를 제거하고 각 잘하는 염료 라벨 용지 200 μl를 추가합니다. 37 샘플을 품어 ° C 5 % CO 30 분 2. </li><li> 전체 F - 12K 10 ML에, 샘플 잠복기 있지만, Hoechst 33342, trihydrochloride, trihydrate (Invitrogen 분자 프로브 물 10 MG / ML 솔루션, 16.2 MM 솔루션) 1 μl를 혼합하여 시료의 핵 얼룩에 대한 Hoechst 33342 용액을 준비 . </li><li> 모든 샘플에서 미디어를 제거하고 각 샘플에 희석 Hoechst 솔루션 600 μl를 추가합니다. 37 샘플을 품어 ° C, 5 % 5 분 동안 CO 2. </li><li> F - 12K 전체 600 μl와 샘플을 세 번 씻으십시오. </li><li> 37 샘플을 품어 ° C 5 % 비법인 형광단이 세포 밖으로 확산 수 있도록 추가 30 분 CO 2. </li><li> 30 분 후에, SNAP 세포 샘플을 마지막으로 한 시간에 미디어를 변경하고 이미지로 넘어갑니다. </li><li> 자이스 혈구 Apotome Axiovert 200M 형광 현미경을 사용하여 이미지 세포. </li></ol> 대표 결과 <img src="/files/ftp_upload/1876/1876fig1.jpg" alt="그림 1" /> 그림 1. 왔지SNAP - 태그 fusions을 표현 아르 라이브 COS - 7 세포의 형광 현미경 표준을 사용하는 형광 이미징의 일부 대표적인 결과입니다. 첫 번째 이미지는 SNAP 세포 TMR - 등급으로 표시 pSNAP - H2B을 (히스톤 핵) 표현 라이브 COS - 7 세포를 보여줍니다. pSNAP - H2B 건설은 pSNAP 태그 (M) 벡터를 사용하여 생성되었습니다. 세포는 30 분 SNAP - 셀 TMR - 스타로 표시하고 핵에 대한 Hoechst 33342 (파란색)과 counterstained되었습니다. 핑크 형광는 히스톤 단백질이 핵을 나타내는 파란색의 형광 이외에 존재하는 적색 형광의 오버레이를 보여줍니다. H2B 단백질의 표현이 명확하고 쉽게 식별됩니다. <img src="/files/ftp_upload/1876/1876fig2.jpg" alt="그림 2" /> <img src="/files/ftp_upload/1876/1876fig3.jpg" alt="그림 3" /> 그림 2와 3.이 다음 두 이미지는 transiently pSNAP - ADRβ2와 transfected 라이브 COS - 7 세포를 보여줍니다. 전지는 SNAP 세포 TMR - 스타 (적색)으로 표시하고 Hoechst 33342 (파란색)과 counterstained되었습니다. pSNAP - ADRβ2 건설은 pSNAP 태그 (M) 벡터를 사용하여 생성되었습니다. 세포는 30 분 SNAP - 셀 TMR - 스타로 표시하고 핵에 대한 Hoechst 33342 (파란색)과 counterstained되었습니다. 파란 형광이 핵을 식별하는 동안 빨간색 형광은 세포 표면 수용체 단백질 ADRB2의 존재를 나타냅니다.

<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>

저자는이 문서에서 사용되는 시약 및 악기를 생산하는 뉴 잉글랜드 Biolabs 고용하고 있습니다.

형광단의 photostability은 퇴색하기 전에 캡처해야합니다 얼마나 빨리 형광 결정 등 초점, 레이저 강도, 장비 기능, 렌즈 사용 등 관찰 결과에 영향을 미치는 여러 변수가 있습니다. 단백질 라벨의이 방법은 다양한 여러 애플 리케이션에 적합합니다. 합성 프로브와 함께 융합 단백질의 특정 상표에 대한 SNAP - 태그와 클립 태그 기술은 라이브 세포에서 세포 lysates의 역동적인 프로세스를 포함하여 다양한 단백질 기능의 다양한 측면을 활성화하십시오. SNAP -, 클립, MCP 및 ACP 태그 기술이 생활하고 고정된 세포에 단백질의 이미지와 체외에서 단백질 연구에 단순하고 특별한 다기능을 제공합니다. 하나의 유전자 구조의 생성은 fluorophores, 비오틴, 또는 구슬을 포함하여 기능 그룹의 다양한 공유 결합 결합을 형성 수있는 태그 융합 단백질을 산출. 관심의 단백질은 복제 및 표현 한 번만 후 융합이 같은 라이브 셀 내부에 동시 단백질 라벨, 단백질 현지화 및 translocation, 펄스 - 체이스 실험, 수용체의 국제화 연구 등 다양한 스트림 애플 리케이션을위한 형광 기판의 다양한 사용할 수 있어야합니다 , 선택적 세포 표면 라벨, 단백질은 assays, SDS - PAGE, 유동세포계측법, microtiter 접시, 바이오 센서 상호 작용 실험에서 높은 처리량 바인딩 assays의 단백질 감지, 그리고 걱정 기반 바인딩 assays를 가져옵니다.

<!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 - 태그와 클립 태그 단백질 라벨링 시스템은 라이브 세포에 대한 관심의 단백질, 형광 염료를 포함하여 분자의 특정 공유 결합 첨부 파일을 활성화합니다. 이러한 시스템은 이미징 장비의 범위에 최적화된 형광 기판의 넓은 선택을 제공합니다. 일단 복제된과 표현, 태그 단백질 다시 복제하지 않고도 수많은 스트림 애플 리케이션을위한 기판의 다양한 사용할 수 있습니다. 이 시스템을 사용하는 두 가지 단계가 있습니다 : SNAP - 태그 융합, 그리고 선택의 SNAP 태그 기판과 융합의 상표로 관심의 단백질의 복제, 그리고 표현. SNAP 태그 인간 O를 기반으로 작은 단백질이다<sup&gt; 6</sup(hAGT)&gt; - alkylguanine - DNA - alkyltransferase DNA 수리 단백질. SNAP 태그 레이블은 벤질 링커를 통해 구아닌에 복합 염료 또는 chloropyrimidine 떠나는 그룹입니다. 라벨 반응에서 기판의 치환 벤질 그룹은 covalently SNAP 태그에 첨부되어 있습니다. CLIP - 태그 benzylcytosine보다는 benzylguanine 파생 상품과 함께 반응하도록 설계 SNAP 태그의 수정된 버전입니다. SNAP 태그와 함께 사용할 경우, 클립 - 태그는 동일한 셀에 동시에 두 단백질의 직교 및 보완 라벨 수 있습니다.

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 - 태그와 클립 태그 단백질 라벨링 시스템은 라이브 세포에 대한 관심의 단백질, 형광 염료를 포함하여 분자의 특정 공유 결합 첨부 파일을 활성화합니다. 일단 복제된과 표현, 태그 단백질 다시 복제하지 않고도 수많은 스트림 애플 리케이션을위한 기판의 다양한 사용할 수 있습니다.

COS - 7 형광 라벨링는 라이브 셀 이미징에 대한 SNAP - 태그 퓨전 단백질 표현

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 조회수.  New England Biolabs. SNAP - 태그와 클립 태그 단백질 라벨링 시스템은 라이브 세포에 대한 관심의 단백질, 형광 염료를 포함하여 분자의 특정 공유 결합 첨부 파일을 활성화합니다. 일단 복제된과 표현, 태그 단백질 다시 복제하지 않고도 수많은 스트림 애플 리케이션을위한 기판의 다양한 사용할 수 있습니다.

비디오: 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.

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

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 ...

연구

생물학

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).

형광등을 통해 F - 굴지 역학 라이브 셀 이미징은 현미경을 스페클 (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.

의 형광 라벨링 Drosophila 심장 구조

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.

의 이식 GFP - 표현 키메라 Zebrafish의 태아의 망막과 두뇌 개발의 라이브 영상에 대한 Blastomeres을

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.

라이브 세포에 형광 단백질과 듀얼 프로브 광학 하이라이트 기능을 정화의 Photoconversion

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.

DNA의 손상 응답 단백질의 2와 3 차원 라이브 셀 이미징

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.

에 GFP-라벨 단백질의 영상을 라​​이브<em&gt; Drosophila</em&gt; Oocytes

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.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

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.

형광 단백질을 표현 라이브 마우스의 이미징 세포 이하의 구조물의 intravital 현미경

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.

Murine 골 수 유래 수지상 세포와 대 식 세포에 형광 성 융해 단백질의 표정

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