우리는 갇힌 플루오레신 만들기에 대한 도움을 댄 칼린 감사하고 싶습니다. 또한 우리는 다음과 자금 출처 감사의 말씀을 전합니다 : JAC에 발달 생물학 교육 그랜트를 위해 밴더빌트 대학 메디컬 스쿨 프로그램을, 그리고 NIH HD054534to JTG.

1. 갇힌 플루오레신 Dextran의 합성 <ol><li> 3.5 밖으로 측정 - aminodextran 4 MG과 CMNB - 갇힌 플루오레신 SE 1 MG를 포함 Invitrogen - 공급 적용된 튜브에 추가합니다. 우리 손에이 비율은 dextran 당 ~ 2.5 염료 분자의 평균 로딩을 제공합니다. </li><li> 튜브에 0.1 M 나 2 B 4 O 7 (나트륨 borate) 버퍼 500 μL를 추가합니다. </li><li> aminodextran와 갇힌 플루오레신를 해산하기 위해 30 초 동안 모자 와동. </li><li> vortexing 믹서에서 하룻밤 반응하자. </li><li> Zeba의 스핀 칼럼의 맨 아래에 폐쇄를 트위스트 후 뚜껑을 느슨하게. 느낌 - 팁 펜으로 열에서 점이 표시합니다. 15 ML 원뿔 튜브에 넣어 칼럼. </li><li> 회전자의 중심을 향하게 점 2 분 1,000 XG에 원심 분리기. 바로 압축 후 열을 사용합니다. </li><li> 새로운 15 ML 원뿔 튜브 압축 열의를 배치합니다. </li><li> 풀 반응 혼합물과 압축 열 수지 침대 센터로 전송할 수 있습니다. 뚜껑을 교체합니다. </li><li> 회전자의 중심을 향하게 점 2 분 1,000 XG에 원심 분리기. 스핀 후 eluent과 칼럼의 맨 위에는 약 색상 노란색 같은 그늘 것입니다. </li><li> 1.5 ML의 microcentrifuge 관에 노란색 eluent (~ 350 μL)를 전송합니다. </li><li> speedvac에서 건조하는 Lyophilize. 호일로 speedvac 표지. </li><li> 1% W / V.의 최종 농도로 물에 옅은 노란색 거품의 다시 중단 결과 ~ 2-3 MG </li><li> 호일 덮인 튜브에서 -20 ° C에서이 솔루션을 저장합니다. 그것은 나누어지는을 반복 냉동 해동을 방지하기 위해 dextran 솔루션 것이 좋습니다. </li></ol> 2. Zebrafish의 태아으로 갇힌 플루오레신 Dextran의 분사 <ol><li> 0.2M KCl에 갇힌 플루오레신 dextran 1시 5분 또는 1시 10분의 1 % W / V 주식을 희석. </li><li> 0.5을 주사 - 압력 주입기를 사용하여 세포 하나의 단계 배아의 노른자에 갇힌 플루오레신 솔루션 1.0nL. 일단 주입, 배아의 클러치는 ° C가 아닌 특정 uncaging을 줄이기 위해 28.5의 어둠 속에 보관해야합니다. </li><li> 수동으로 배아에서 chorions를 제거합니다. </li><li> 4퍼센트 tricaine에서 배아를 마취. </li><li> 배아는 다음 달걀 물로 덮여, 35mmx10mm 배양 접시에있는 0.8 % 아가로 오스에 마운트됩니다. </li><li> 적어도 15 분 동안 50 ° C 열 블록에 녹아 아가로 오스를 평형. </li><li> 유리 피펫을 사용하여 물을 최소한 한 배아를 그리기와 아가로 오스 참고로 그것을 릴리스 :. 그것은 피하기 위해 튜브로 배아를 퍼팅하기 전에 약 30 초 동안 손에 아가로 오스의 튜브를 냉각하는 것이 중요합니다 배아를 죽이는. </li><li> 약 50μL 아가로 오스의와 함께, 튜브의 배아를 피펫하고 페트리 접시의 중앙에 내용을 꺼냅니다. </li><li> 신속 동양 최대 직면 uncaged하는 세포와 플라스틱 스레드를 사용하여 배아를 참고. 시원한 신속 때문에 배아를 orienting와 서두르지와 아가로 오스. 아가로 오스는 단단한 된 후 조작하면 태아도 손상될 수 있습니다. </li><li> 아가로 오스가 완전히 고체화 (이 몇 분 소요) 및 4% tricaine와 0.003 %의 PTU를 포함하는 계란 물을 전체 요리 삼분의이을 채울 수 있습니다. </li></ol> 3. 플루오레신 Dextran의 레이저 Uncaging <ol><li> uncaging에 사용되는 현미경 40X 물 침지 목적을 가지고 있어야합니다. </li><li> 우리는 365 나노미터 색소 세포와 70 % / 30 % 분할 이색성 거울 Photonics 인스 트루먼 트의 레이저를 사용합니다. </li><li> 이전 uncaging로, 레이저는 객관적이고 적절한 능력을 감쇠와 parfocal하셔야합니다. </li><li> 레이저 초점을하려면 목표 아래 미러 유리 슬라이드를 삽입하고 슬라이드에 흠집을 볼 때까지 목표를 중점을두고 있습니다. </li><li> 레이저가 단일 펄스와 함께 거울을 명확하게 눈에 띄는 흠집을 생산 수있을 때까지 레이저 감쇠기를 조정합니다. </li><li> 목표의 초점을 조정합니다 아래. 목적 초점 밖으로 때 레이저 흠집을 생산할 수있다면, 레이저 초점을 조정 ¼ 아래 켜거나. 레이저는 목적이 초점을 맞춘 경우에만 거울을 흠집 수있을 때까지 반복합니다. </li><li> 장소 uncaged하는 지역의 목적과 집중에 따라 배아를 탑재. </li><li> 새장에서 내놓다하려면, 관심의 세포에 초점을 후 2-3 Hz에서에서 그것을 10-20 번 펄스. </li><li> 포셉와 멀리 아가로 오스를 필링 계란 물에 퍼팅 0.003 %의 PTU를 포함하여 uncaging 후, 무료로 배아. 그들은 고정 때까지 배아가 빛을 꽉 상자에서 개발하자. </li></ol> 4. 항체 라벨링 및 Uncaged 플루오레신 Dextran의 감지 <ol><li> AB 정착액의 배아 수정 4 실온 (RT) 또는 야간 (O / N)에서 2-4시간 ° C.에 대한 (4 % paraformaldehyde, 0.3mM CaCl 2, 8 % 자당을 1X 인산은 살린, 산도 7.3를 버퍼) 모든 라벨 단계에 대한 가벼운 꽉 상자나 포일 싼 튜브의 배아 보관하십시오. </li><li> 정착액을 제거하고 100 % MeOH로 교체, RT에서 10 분 정도두고, MeOH와 repla를 제거신선한 100 % MeOH로 CE. </li><li> 적어도 30 분 -20 ° C에서 MeOH에 보관주의 :. epitopes가 저하하기 시작하기 전에 당신이 최대 두 주 동안 MeOH에 저장할 수 있습니다. </li><li> 75% MeOH/1X 인산의 RT에서 오분 세척과 배아를 Rehydrate은 0.1 % 트윈 20 (1X PBSTw)와 생리 버퍼 후 50% MeOH/50 %의 1XPBTw 다음 25% MeOH/75 % 1X PBSTw. </li><li> 1XPBSTw 5 분 세 번 씻으십시오. </li><li> 배아는&gt; 24 시간 전거있다면, 10 μg / 1XPBSTw에 ML proteinase K와 배아를 permiabilize. Permiabilization 시간은 무대에 따라 달라집니다. 배아는 24 48시간 후 수정 (HPF) 사이하는 경우 proteinase K에 5 분 정도면 충분합니다. 더 많은 시간이 오래된 애벌레에 대한 경우 필요할 수 있습니다. RT 20 분 AB 정착액에 다시 수정 </li><li> RT에서 1XPBSTw 5 분 다섯 번 씻으십시오. </li><li> RT에서 1-2시간에 대한 (1XPBS, 0.1 %의 트리톤 x100 10 % 양 혈청 10 % 소 혈청 횐자위, 1 % 디메틸 sulfoxide) 블록 솔루션에 품어. </li><li> 솔루션을 차단의 기본 항체 (안티 플루오레신 플러스 셀 타입의 특정 마커에 대한 항체)를 희석. </li><li> 4 품어 배아 O / N ° 일차 항체를 포함하는 솔루션에 C. </li><li> 1XPhosphate에 20 분 네 번 씻으면 RT에서 0.1 %의 트리톤 x100 (PBSTx)와 살린 버퍼. </li><li> 솔루션을 차단에 찬란 - 레이블 차 항체 (대개 1:300) 희석. </li><li> 4 품어 O / N ° 보조 항체를 포함하는 솔루션에 C. </li><li> RT에서 1XPBSTx에 20 분 네 번 씻으십시오. </li><li> RT에 적어도 두 시간 50 % glycerol/50 %의 1XPBS에 맑은 배아는 다음 글리세롤 / PBS 혼합물을 제거하고 100 % 글리세롤을 추가하고 4 O / N 서하자 ° C.를 </li></ol> 5. 대표 결과 : immunolabeling 후 (Figure.1) uncaged 있던 세포에 얼룩의 풍부한 공간이 있어야합니다. 그것은 플루오레신의 자연 uncaging로 인해&gt; 48시간 이전 zebrafish에서 잡음 비율로 비교적 높은 신호를 볼 수 드문되지 않습니다. <img src="/files/ftp_upload/2672/2672fig1.jpg" alt="그림 1" /> 그림 1. zebrafish 뇌의 복합 리니지 라벨. 24 HPF, foxd3로 표시 앞쪽에 뇌의 anlage의 일부, :. GFP 표현이, UV 레이저 펄스를 사용하여 uncaged했습니다)이 48hpf함으로써, 세포의 왼쪽 단면 클러스터 parapineal라고 불리는 (빨간색 동그라미) uncaged에서 등장했습니다 도메인 (화이트 서클). B)는 Uncaged 플루오레신 신호 parapineal (빨간색 동그라미의 강화)과 뇌의 기관 (흰색 동그라미).입니다

<ol>
	<li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+the+epiblast+of+the+early+chick+embryo.">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina.">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+fluorescent+tracers+to+study+neural+cell+lineages.">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , 1-8 (1991).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li><li>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuC:Kaede,+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo.">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li><li>Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101, 15905-15910 (2004).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Chemicals. , 707-711 (2002).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+(caged)+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo.">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+first+cleavage+in+the+sea+urchin+embryo,+Lytechinus+variegatus,+does+not+specify+the+axes+of+bilateral+symmetry.">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li></ol>

관심 없음 충돌 선언하지 않습니다.

설명한 바와 같이,이 프로토콜은 microinjection, 현미경, 그리고 immunofluorescence의 일반적으로 사용되는 기법을 기반 zebrafish에서 상대적으로 빠른 가계 라벨 방법을 제공합니다. 우리는 레이저화된 방식으로 플루오레신을 개방하다 가장 효율적이고 비용 효과적인 방법으로 uncaging 것으로 나타났습니다. 이 방법은 4 DPF 늦게 같은 실험 끝점과 혈통의 라벨을 사용할 수 있습니다. 그러나, 세포가 분열로 ...

A correction was made to <a href="http://www.jove.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CMNB-caged fluorescein SE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C20050</td>
<td>&nbsp;</td>
<tr>
<td>Aminodextran, 10kDa</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1860</td>
<td>&nbsp;</td>
<tr>
<td>Zeba desalt spin columns</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>89889</td>
<td>&nbsp;</td>
<tr>
<td>goat anti-fluorescein antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A11095</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 633 Donkey anti-goat antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A21082</td>
<td>&nbsp;</td>
<tr>
<td>35mmx10mm petri dishes</td>
<td>&nbsp;</td>
<td>Becton-Dickinson</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Upright compound microscope with 40x water immersion objective</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DM6000B</td>
<td>&nbsp;</td>
<tr>
<td>MicroPoint Manual Laser Illumination System</td>
<td>&nbsp;</td>
<td>Photonic Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Alfa Aesar</td>
<td>L06690</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E10521</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115836991</td>
<td>&nbsp;</td>
<tr>
<td>Plastic thread</td>
<td>&nbsp;</td>
<td>Any sewing supply store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Research Products International</td>
<td>A20090</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>Savant SPD131DDA</td>
<td>&nbsp;</td>
<tr>
<td>Vortexing mixer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>Vortex Genie 2</td>
<td>&nbsp;</td>
<tr>
<td>Pressure injector</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>MPPI-3</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

발달 생물학의 중심 문제는 그들이 undifferentiated 엽 성의 전구 물질로부터 발생하는 등 척추 동물에있는 무수한 세포 유형의 출처를 알아낼 것입니다. 연구원은 DiI 상표 1 및 모델 시스템의 개발의 나중 단계에서 확인할 세포 운명을 추적 효소의 압력 주입이 같은 혈통 라벨의 다양한 방법을 고용했습니다. zebrafish (Danio rerio)의 첫 번째 운명의지도는 배아의 이산 지역에서 하나의 세포로 같은 rhodamine dextran과 같은 형광 염료의 주입에 의해 iontophoretic 조립 시간 동안 3-5 라벨 세포의 운명을 추적했다. 효과적인 반면,이 방법은 기술적으로 요구하고 일반적으로 zebrafish 실험실에서 찾을 수 없습니다 전문 장비가 필요합니다. 최근, 이러한 자외선에 노출되면 irreversibly 빨간색 형광 초록색에서 전환 에오스와 가에데로 photoconvertable 형광 단백질은, zebrafish 6-8 증가 사용을 볼 수 있습니다. zebrafish 배아와 transgenesis의 상대적 용이성의 광학 선명도는 혈통의 라벨에 대해 이러한 특히 매력적인 도구를 만들었습니다 그리고 생체내 7 세포의 마이 그 레이션을 관찰합니다. 그들의 유틸리티에도 불구하고,이 단백질은 염료 중재 혈통의 라벨 방법 몇 가지 단점을 비교합니다. 가장 중요한 우리는 이러한 단백질 photoconversion 동안 높은 3 - D 해상도를 얻기에서 발견 어려움이다. 이 빛 가운데, 아마도 zebrafish의 혈통 라벨에 대해 해상도와 사용 용이성에있어 최고의 조합 갇힌 플루오레신 dextran의 사용, 칭 그룹에 바인딩된 형광 염료를 만드는 마스크는 형광 9. 염료 다음은 형광 또는 면역의 시각화를 허용, 레이저 또는 수은 램프에서 자외선을 사용하여 특정 셀 내의 (담금질 그룹에서 발표) &quot;uncaged&quot;가 될 수 있습니다. iontophoretic 방법과는 달리, 우리에 갇힌 플루오레신은 표준 사출 apparatuses로 주입 수 있으며 핀홀 10 갖춘 epifluorescence 현미경으로 uncaged. 또한, 플루오레신에 대한 항체는 uncaged 양식을 감지하고, 에피토프는 고정 잘 남아 11. 마지막으로, 우리에 갇힌 플루오레신는 두 개의 광자 현미경은 12,13 고용 특히 경우, 매우 높은 3 - D 해상도 활성화할 수 있습니다. 이 프로토콜은 갇힌 플루오레신 및 레이저 uncaging에 의해 혈통 라벨의 방법을 설명합니다. Subsequenctly, uncaged 플루오레신는 항체로 분류하여 같은 GFP와 같은 다른 epitopes와 동시에 검색됩니다.

Watch this Scientific Journal Video about Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran at JoVE.com

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

이 프로토콜은 uncaged 플루오레신에서 신호를 증폭하기 위해 immunolabeling 전체 산 뒤에 갇힌 플루오레신의 UV - uncaging를 사용하여 셀 zebrafish의 배아의 작은 그룹의 운명을 라벨 및 추적하는 방법을 delineates.

레이저 Uncagable 플루오레신 Dextran과 Zebrafish 세포의 리니지 라벨

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

lineage-labeling-zebrafish-cells-with-laser-uncagable-fluorescein

Research

JoVE Journal

Biology

13.8K 조회수.  Vanderbilt University. 이 프로토콜은 uncaged 플루오레신에서 신호를 증폭하기 위해 immunolabeling 전체 산 뒤에 갇힌 플루오레신의 UV - uncaging를 사용하여 셀 zebrafish의 배아의 작은 그룹의 운명을 라벨 및 추적하는 방법을 delineates.

비디오: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

신진 대사 라벨링 및 Bioorthogonal 클릭 화학에 의해 Zebrafish 태아의 영상 Glycans

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Zebrafish의 Pronephros의 레이저 절제는 신장 상피 조직이 재생된를 공부

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

지질 뗏목의 결정은 형광 상관 분광학 (FCS)에 의해 살아있는 세포에서 Fluorescently 태그가 추가된 프로브의 파티션

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Murine 조혈 줄기 세포와 리니지 - 헌신 Progenitors의 Phenotypic 분석 및 절연

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebrafish 배아 골격근의 레이저 쏜 부상

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

하나 나노 미터의 정확도와 형광 이미징 (피오나)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

시각화 및 간 엽 세포 계보 특정 표시와 함께 Adipogenic 차별화 가능성의 정량화

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

이중 마커를 이용한 모자이크 분석을 이용한 대뇌 피질 개발의 계보 추적 및 클론 분석(MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...