JB는 데몬 런연 암 연구 재단 원정대 DRG - 1716-02에 의해 지원되었다. 점프 온 제로는 NIH / NIGMS 원정대 F32 GM082174 - 02에 의해 지원되었다. NP는 하워드 휴즈 의학 연구소의 수사관이다.

1. 파리 케이지의 준비 <ol><li> 같은 32 온스의 곤충 컵 등 편리한 용기 Drosophila (예 : 오레곤 - R 또는 유전자형과 같은 야생 형 변종) 성장. </li><li> 대형 배아 - 수집 연습장 새로 eclosed 파리를 전송하거나 인구 새장을 날아. </li><li> ~ 25의 온도와 공간에 새장을 이동 ° C 습도 65 ~ 12 H 비추어 %와 동기식 태아의 수집을 용이하게하기 위해 검은 12 H 사이클. </li><li> 그들 몰레 시즈 접시에 줄무늬 효모 붙여넣기를 죽인 먹이로 새장에 파리를 유지합니다. 2-3를위한 하루에 한 번 당밀 번호판을 (죽은 효모 붙여넣기)를 변경 D. </li></ol> 2. 동기 배아 수집 <ol><li> 기본 셀 준비의 날, 1-2 H 미리 누워 기간 동안 붙여 살해 효모와 코팅 파리 wth 새로운 번호판을 공급. </li><li> 단지 12 - H 어두운주기 전에 효모 붙여넣기의 최소 금액과 함께 줄무늬 신선한 당밀 플레이트와 함께 사전에 누워 접시를 교체하십시오. 비동기 이전 배아를 포함하는 사전 누워 컬렉션을 폐기. </li><li> 1-2 H. 기간 동안 태아를 수집 </li><li> 5-6 H. 25에서 배아와 저장소의 비교적 동기 인구를 포함하는 접시 ° C를 제거 배아 이번에 고급 gastrula 무대에있을 것입니다. </li></ol> 3. 수집 및 배아의 세척 <ol><li> 배아는 하단에있는 최고급 메쉬 체에서 수집하므로, 젖은 페인트와 함께 접시에서 배아를 풀어, 그리고 sieves의 스택 집합을 통해 미지근한 수돗물로 깨끗이 씻어. 가능한 한 많은 효모 및 비행 파편으로 제거하는 몇 분 동안 씻어. </li><li> 팁 배아는 한쪽에 축적되도록 sieves, 그리고 부드럽게 dechorionation 바구니에 체에서 그들을 씻어. </li><li> 배아가 건조 아무말도없이 조직 문화 후드 영역으로 이동합니다. </li></ol> 4. 배아의 균질화 및 세포의 도금 <ol><li> 소독 50 % (권 / 권) 50-10 분 표백제에서 그들을 immersing하여 배아를 d​​echorionate. 70% (권 / 권) 에탄올과 바구니의 벽에서 배아를 씻어. 또는 에탄올 치료를 받기 전에 증류수로 배아에서 표백제를 씻어. 이 시점에서 온워드 배아는 무균 조건과 조직 문화 후드에서 처리되어야합니다. </li><li> autoclaved 증류수와 표백제 및 / 또는 에탄올을 제거 철저하게 배아를 씻어. </li><li> dechorionation 단계 동안, 태아의 크기에 따라 실내 온도 M3 매체의 적절한 볼륨 다운스의 균질 화기를 입력하십시오. 배아의 볼륨 사이와 미디어의 비율은 0.5 - ML 배아 이상해서는 안됩니다 : 40 - ML 미디어 (또는 ~ 100-200 배아 / ML). </li><li> 잠수함 배아를 충분히 M3 미디어의 금액과 소독을 비커에 씻어서 배아를 놓습니다. 배아는 2-5 분 흠뻑 젖어 보자. </li><li> dechorionation 바구니를 분해하고 소독을 종이 타월로 Nitex 메쉬가 건조 얼룩. </li><li> 멸균 페인트로 균질 화기로 배아를 전송합니다. </li><li> 모든 배아가 일시 중지 때까지 튜브의 바닥에 도달하지 않고 느슨한 유봉을 사용하여 짧은 스트로크와 함께 부드럽게 균질. 부드럽게,하지만 단호하게, 여덟 전체 스트로크와 균질. 그것이 아래로 이동하고 같은 유봉을 비꼬아하지 마십시오. </li><li> 용탕 주입이나 pipetting을 통해 살균 50 ML 원뿔 원심 튜브에 homogenate를 전송하고, 튜브 캡. </li><li> 40g, 펠렛을 실온 조직 파편, 큰 세포 대단히 짧은 시간과 vitelline의 점막에서 10 분 원심 분리기. 깨끗한 튜브에 세포와 노른자가 들어있는 뜨는을 전송 5 분 원심 분리 단계를 반복합니다. </li><li> 세포를 따르고 또는 펠렛으로, 360g 10 분 깨끗한 튜브와 스핀으로 실내 온도를 pipetting하여 표면에 뜨는를 주의깊게 전송합니다. 뜨는 폐기하십시오. 문화 매체 10 ML에있는 세포 펠렛을 (RNAi 실험을위한 혈청 무료 중간) Resuspend. </li><li> 세포 밀도를 추정 hemocytometer를 사용하여 가능한 셀을 계산합니다. 한편, 펠렛 세포에 4.10 단계를 반복합니다. </li><li> 원하는 농도로 세포를 Resuspend. 시작하려면 1의 범위 ~ 5 X10 6 셀 / ML 및 1.7-2.5 X10 5 셀 / cm 2 판 밖으로보십시오. </li></ol> 5. 384 잘 접시에 성장 세포에 대한 기본 셀 RNAi <ol><li> 384 잘 플레이트에 잘 당 ~ 250 NG의 최종 농도 각 우물에 dsRNAs 5 μl를 분배. </li><li> 멀티 채널 피펫을 사용하여, 각 우물에 다른 dsRNA를 포함하는 384 잘 플레이트에 혈청 무료 M3 매체에 잘 따라 기본 셀 10 ML (1 ~ 4x106 세포 / ML)를 분배. 또한, 전지가 공촛점 이미징 8 - 음 유리 챔버 슬라이드에서 교양 수 있습니다. </li><li> 300g, 실온에서 1 분 번호판을 원심 분리기. 25 혈청없는 매체 문화 세포 ° 22 H 또는 18 ° C 1-2위한 C D. </li><li> 각 잘하는 혈청 함유 배지 30 ML을 추가합니다. </리&gt; <li> 300g, 실온에서 1 분 번호판을 원심 분리기. </li><li> ~ 7 D 25 추가 5 습기 챔버 문화 기본 세포 ° C.에 접시를 놓고 </li><li> 이미지 세포도 살지 immunofluorescence 염색법 후. </li></ol> 6. 대표 결과 : 최적의 조건에서, 문화에있는 세포 둥근 형태로 건강을 찾습니다. 문화 작은 세포 파편을 가지고 있고, 초기 도금 후 50 ~ 80 % 합류한다. 이러한 세포는 오랜 기간 동안 교양 후 형태학의 변화를 받아야합니다. 그것은 일반적으로 완벽하게 25 차별하는 문화의 세포 약 5~8일 소요 ° C. 좋은 품질의 차 문화는 일반적으로 세포 수준의 관심이 문화에 차별하는 방법 잘으로 판단됩니다. 예를 들어, 기본 근육 세포들은 종종 지점과 같은 형태를하고 sarcomeres 일련의 구성 myofibrils 같은 줄무늬가있는 multinucleated myotubes된다. 완전 차별 myotubes은 대개 저절로 문화에 이동합니다. 대조적으로, 차별화된 기본 뉴런들은 세포 기관과 클러스터를 형성하고, 그들의 축삭 케이블 다른 클러스터와 연결합니다. <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="그림 1" /> 그림 1. 기본 세포는 문화에 도금 후 morphologic 변경 사항을 받다 오른쪽 384 잘 판의 우물에 도금 후 기본 셀 (A) 명시야 이미지. 세포의 대부분은 원형 그대로 잘 구분됩니다. 기본 세포 배양의 (B) 명시야 이미지 20시간 도금 후. 차 전지는 이미 지점과 같은 형태 (긴 화살표)와 클러스터 (큰 애로)와 근육이 단계에서 인식 수를 형성하고 있습니다. 오일 도금 후 주요 문화 (C) 위상 대비 영상. 문화의 연결을 확장 (작은 화살표)의 연결을 클러스터 (대형 화살촉)와 근육 (중소 화살표)와 함께 더 많은 고급 보인다. <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="그림 2" /> 그림 2. dsRNAs 취급 차 전지의 예 384 잘 판에 교양 차 전지는 Dmef2 - Gal4 들고 배아, D42 - Gal4, UAS - 미토 - GFP의 transgenes,하는 Dmef2 - Gal4 및 근육과 운동 신경에 미토 - GFP의 D42 - Gal4 드라이브 표현, 각각에서 고립되었다. 세포 lacZ (AC) 또는 비정상적으로 증가 (경우) (DF) 중 하나를 대상으로 dsRNAs로 치료했다. 주요 근육과 신경 모두 GFP (A, C, D, F)로 볼 수 있습니다. 흰색 화살표의 연결을 확장을 가리 킵니다. 굴지 (화살촉)의 Phalloidin의 얼룩은 lacZ dsRNAs (B와 C) 대우 문화에 지점과 같은 근육 구조를 공개하지만, 문화에 라운드 - 업 근육 형태가있다면 함께 치료 dsRNAs (E와 F). 병합된 이미지 (C, F)에서 근육이 노란색 스테인드지만, 붉은 부정적인 쇼 뉴런과 확장 (흰색 화살표). 아르 <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="그림 3" /> 그림 3. 주요 근육의 공촛점 micrographs 인간 vitronectin - 코팅 커버 유리 슬라이드에 교양 주요 근육은 immunofluorescently 방지 Drosophila Titin (KZ) (병합에 빨간색)과 굴지에 대한 phalloidin (병합에 녹색) 물들일되었습니다. 상단 패널은 야생 형 제어 근육이며, 아래 것들 살스 (CG31374) 단축 sarcomeres와 RNAi의 근육입니다.

<ol>
	<li>Ashburner, M., Roote, J., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory culture of Drosophila. , 588-599 (1999).</li><li>Bai, J., Hartwig, J. H., Perrimon, N. S. A. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+WH2-domain-containing+protein,+promotes+sarcomeric+actin+filament+elongation+from+pointed+ends+during+Drosophila+muscle+growth.">a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth.</a> Dev. Cell. 13, 828-842 (2007).</li><li>Bai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+Drosophila+primary+cells+for+genes+involved+in+muscle+assembly+and+maintenance+.">RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance .</a> Development. 135, 1439-1449 (2008).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PloS. Genet. 4, e1000111-e1000111 (2008).</li><li>Donady, J. J., Fyrberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+culturing+of+Drosophila+embryonic+cells+in+vitro.">Mass culturing of Drosophila embryonic cells in vitro.</a> TCA Manual. 3, 685-687 (1977).</li><li>Seecof, R. L., Alleaume, N., Teplitz, R. L., Gerson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+neurons+and+myocytes+in+cell+cultures+made+from+Drosophila+gastrulae.">Differentiation of neurons and myocytes in cell cultures made from Drosophila gastrulae.</a> Exp. Cell Res. 69, 161-173 (1971).</li><li>Bai, J., Sepp, K. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+primary+cells+dissociated+from+gastrula+embryos+and+their+use+in+RNAi+screening.">Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.</a> Nature Protocols. 4, 1502-1512 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Drosophila 세포의 주요 문화의 발달 패턴은 크게 기본 셀 준비 과정에서 여러 변수에 따라 가능 다를 수 있습니다. 우선, 달걀 부설에 사용되는 파리 젊은이가 (이전 일주일 이내)과 건강 (바이러스 또는 세균 감염은 무료). 있어야 그들이 쓸모 있고 그 배아에서 추출한 세포가 구별 수없는 오직 1~2일 위해 살아남을 것이다로 Unfertilized 또는 감염된 태아는 피해야한다. 둘째, 균질화 과정에서, ?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Shields and Sang M3 medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>JRH Biosciences</td>
<td>12103-500M</td>
<td>&nbsp;</td>
<tr>
<td>Insulin from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-6634</td>
<td>&nbsp;</td>
<tr>
<td>Large embryo collection cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-101</td>
<td>&nbsp;</td>
<tr>
<td>#25 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-454</td>
<td>&nbsp;</td>
<tr>
<td>#45 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-462</td>
<td>&nbsp;</td>
<tr>
<td>#120 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-474</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder, Wheaton 7 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>62400-620</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0040</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0100</td>
<td>&nbsp;</td>
<tr>
<td>Costar, 384-well plate</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29444-078</td>
<td>&nbsp;</td>
<tr>
<td>Lab-Tek II Chambered coverglass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>171080</td>
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primary cell cultures from drosophila gastrula embryos

여기 Drosophila gastrula의 배아에서 dissociated을 준비하고 culturing 기본 세포에 대한 방법을 설명합니다. 간단한에서는 많은 양의 젊고 건강한 파리에서 배아를 개최는 수집 소독 및 유리 균질 화기를 사용하여 단일 세포 현탁액에 다음 물리적 dissociated 있습니다. 문화 매체에 적절한 밀도에 문화 접시 또는 챔버 슬라이드에 도금 후,이 세포가 추가로 특정 세포 마커에 의해 확인할 수 있습니다 몇 가지 morphologically - 서로 다른 세포 유형으로 구별하실 수 있습니다. 또한, 우리는 유전자 최저를 이끌어내는 두 번 좌초 (DS) RNAS 이러한 세포를 치료에 대한 조건을 제시한다. Drosophila 차 전지의 효율적인 RNAi는 dsRNA 간단하게 함유 배지에서 세포를 입욕에 의해 수행됩니다. 다른 분자, 생화학, 세포 이미징 분석과 함께 효과적인 RNAi의 섭동을 수행하는 능력은 질문의 다양한 특히 Drosophila 차 전지, 차별화된 근육 세포의 연결에 관련된 사람에 답변 수 있습니다.

Watch this Scientific Journal Video about Primary Cell Cultures from Drosophila Gastrula Embryos at JoVE.com

Primary Cell Cultures from Drosophila Gastrula Embryos

우리는에서 dissociated 기본 세포를 준비 상세한 프로토콜을 제공합니다<em&gt; Drosophil</em&gt; 배아. 다른 분자 생화학 및 세포 이미징 방법과 함께, 효과적인 RNAi의 섭동을 수행하는 능력은 질문 다양한에서 해결될 수 있도록<em&gt; Drosophila</em&gt; 기본 세포.

에서 기본 셀 문화 Drosophila Gastrula의 태아

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

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Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

13.8K 조회수.  Harvard Medical School. 우리는에서 dissociated 기본 세포를 준비 상세한 프로토콜을 제공합니다 Drosophil 배아. 다른 분자 생화학 및 세포 이미징 방법과 함께, 효과적인 RNAi의 섭동을 수행하는 능력은 질문 다양한에서 해결될 수 있도록 Drosophila 기본 세포.

비디오: Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

늦은 단계 Drosophila Pupae의 두뇌에서 기본의 연결을 문화

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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생물학

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Midgastrula 스테이지 Drosophila의 태아에서 문화의 연결 준비

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

쥐 Neonates에서 기본 Dissociated Midbrain의 도파민 세포 문화

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

조직 타겟 배아 Chimeras : Zebrafish Gastrula 세포 이식

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

설치류에서 기본 성인 Fibroblast 문화를 구축

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

의 입식 이미징 Drosophila 태아

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

생활의 이미징 셀 모양 변경 Drosophila 태아

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin의 분리 및 정제에서 Drosophila 태아

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...