우리 감사 합니다 T. Fritsch, A. 늑대-Asseburg, I. Linde와 미-M. 우수 동물 관리 및 기술 지원에 대 한 Tokarski입니다. 우리는 강렬 하 고 유용한 토론에 대 한 Köster 연구소의 모든 구성원에 게 감사입니다. 우리는 기꺼이 도이치 가운데 (코 1949/5-1)와 Niedersächsisches Vorab (VWZN2889) 저 색 소니의 연방 정부에 의해 자금을 인정 합니다.

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저자는 공개 없다.

여기, 우리의 현재 두 개의 서로 다른 프로토콜 문화 1 차 셀 2 dpf zebrafish 태아 또는 성인 zebrafish 두뇌에서.
1 차 셀 문화 2 dpf zebrafish에서 준비 기본 셀 문화 기술에 경험을 가진 사람에 대 한 수행 상대적으로 쉽습니다. 그러나, 좋은 하 고 재현 가능한 결과 얻으려면 시작 물자로 배아의 충분 한 수는 중요 한 (100은 최소). 배아의 발생 하는 동안 모든 가능한 소스 오염 세균과 기...

제 브라 (Danio rerio, D. rerio) 수많은 분야 기본 및 생물 의학 연구1의 척 추가 있는 인기 모델 이다. Zebrafish 태아 utero 전빠르게 개발, 투명 한, 그리고 따라서 살아있는 유기 체에서 척추 개발을 공부 하 고 우수한 필수 구성 요소를 제공 하는 현미경 적합. Zebrafish2의 유전자 추적성 인해 많은 안정적인 유전자 변형 기자 라인 셀 형식 관련 표현의 다양 한 형광 마커 특정 세포 인구의 관찰에 대 한 수 있도록 설립 되었습니다. Zebrafish 커뮤니티 제공 합성 Kal4TA4 (또는 KalTA3에 해당 GalFF) 표현 transgene 수행 소위 Gal4 드라이버 라인의 광범위 한 다양 한 바이러스 transcriptional 활성화 융합 유전자 효 Gal4 DNA 바인딩 도메인 셀 형식 관련 증강의 통제 도메인. 이러한 드라이버 라인 수행 transgenes는 정의 된 상류 활성화 순서 (UAS) 리포터 유전자를 융합의 구성 된 이펙터 라인을 넘어 있다. Kal4TA4 단백질에 따라서 기자 유전자3,4의 셀 형식 선택적 식 활성화 UAS 요소에 바인딩합니다. 이 방법은 거의 모든 사용 가능한 증강 및 기자 요소 이중 유전자 변형 동물의 매우 다양 한 조합 연구에 대 한 수 있습니다.
그러나 개별 셀 또는 그들의 subcellular 내용을 초점 깊이 있는 라이브 영상 전체 하 고 끊임없이 변화 배아에 제한 됩니다. 높은 해상도와 특정 세포 생물학 질문을 해결 하기 위해 세포 배양의 사용은 종종 바람직. Zebrafish의 일부 셀 라인, 하지만 그들은 무 겁 게으로 선택한5,,67 여겨진다 하 그들의 전파는 종종 시간이 걸리는. 또한, 모든 사용 가능한 셀 라인은 구와 파생, 실험 세포 배양을 사용 하 여 셀의 한 종류에 제한. 따라서 우리 모두는 효율적이 고 사용 하기 쉬운 프로토콜 zebrafish 태아 및 접근 문화의 장 수를 증가 하 고 재배의 다양성을 확대 하기 위해 함께 성인 zebrafish 뇌에서 직접 1 차 셀을 준비 하는 설립 셀 유형입니다. 또한, 선물이 형광 세포 기관이 표식에 대 한 식 구문 가진 배아 1 차 셀 transfect에 절차. 따라서, 세포 형태학 및 subcellular 구조 공간 및 시간에서 고해상도 그들의 주요 기능을 유지 하는 다른 세포 유형으로 분석할 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>Fish lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AB (wild-type)</td>
			<td></td>
			<td></td>
			<td>established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC)</td>
		</tr>
		<tr>
			<td>Tg(ptf1a:eGFP)jh1</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the enhancer of the zebrafish gene ptf1a drives expression of the fluorescent protein EGFP (Parsons et al., 2007)</td>
		</tr>
		<tr>
			<td>Tg(XITubb:DsRed)zf148</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the Xenopus neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&#160; (Peri and N&#252;sslein-Volhard, 2008)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>model 5804 R</td>
		</tr>
		<tr>
			<td>ChemiDoc MP imaging system</td>
			<td>BioRad</td>
			<td></td>
			<td>model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos</td>
		</tr>
		<tr>
			<td>confocal laser scanning microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>model SP8, equipped with 28 &#176;C temperature box and a 63 x objective</td>
		</tr>
		<tr>
			<td>epifluorescent microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>model DM5500B, equipped with 28 &#176;C temperature box and a 40 x objective</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell with capacitance extender</td>
			<td>BioRad</td>
			<td>1652661</td>
			<td>electroporation device</td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>GFL</td>
			<td></td>
			<td>model 3011</td>
		</tr>
		<tr>
			<td>incubator for cell culture (28 &#176;C)</td>
			<td>Memmert</td>
			<td></td>
			<td>model incubator I</td>
		</tr>
		<tr>
			<td>incubator for embryos (28 &#176;C)</td>
			<td>Heraeus</td>
			<td></td>
			<td>type B6120</td>
		</tr>
		<tr>
			<td>light microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>model TELAVAL 31</td>
		</tr>
		<tr>
			<td>micro pipettes</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sterile work bench</td>
			<td>Bio Base</td>
			<td></td>
			<td>with laminar flow and UV light</td>
		</tr>
		<tr>
			<td>tweezers</td>
			<td>Dumont</td>
			<td></td>
			<td>Style 5, Inox</td>
		</tr>
		<tr>
			<td>vertical tube rotator</td>
			<td>Labinco B.V.</td>
			<td></td>
			<td>model LD-79</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image Lab Software</td>
			<td>BioRad</td>
			<td></td>
			<td>for the ChemiDoc MP imaging system from BioRad</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016).</td>
		</tr>
		<tr>
			<td>LAS X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>for both confocal and epifluorescent microscopes from Leica Microsystems</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCS-DCX-tdTomato</td>
			<td>K&#246;ster Lab</td>
			<td># 1599</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-eGFP</td>
			<td>K&#246;ster Lab</td>
			<td># 7</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-H2B-mseCFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2379</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-mClover</td>
			<td>K&#246;ster Lab</td>
			<td># 3865</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-MitoTag-YFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2199</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-ss-RFP-KDEL</td>
			<td>K&#246;ster Lab</td>
			<td># 4330</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-VAMP1-mCitrine</td>
			<td>K&#246;ster Lab</td>
			<td># 2291</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pSK-UAS:mCherry</td>
			<td>K&#246;ster Lab</td>
			<td># 1062</td>
			<td>based on the pBluescript-backbone of Stratagene</td>
		</tr>
		<tr>
			<td>Plasmid numbers refer to the database entries of the K&#246;ster lab. Plasmids are available upon request.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plastic and glass ware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Cell Strainer (40 &#181;m)</td>
			<td>FALCON</td>
			<td>REF 352340</td>
			<td>distributed by BD Bioscience, used as &#8220;landing net&#8221; to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium</td>
		</tr>
		<tr>
			<td>1.5 mL reaction tubes</td>
			<td>Sarstedt</td>
			<td>72690550</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL falconic tube</td>
			<td>Sarstedt</td>
			<td>62.547.004</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sarstedt</td>
			<td>83.3924.005</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyStrainer (40 &#181;m)</td>
			<td>Greiner Bio-One</td>
			<td>542 040</td>
			<td>with venting slots; used to filter cells after collagenase-mediated dissociation</td>
		</tr>
		<tr>
			<td>electroporation cuvette (0.4 cm)</td>
			<td>Kisker</td>
			<td>4905022</td>
			<td></td>
		</tr>
		<tr>
			<td>glass coverslips</td>
			<td>Heinz Herenz Medizinalbedarf GmbH</td>
			<td>1051201</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher Scientific (Menzel Gl&#228;ser)</td>
			<td>631-0845</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Henneberg-Sander GmbH</td>
			<td>9020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (plastic; 3 mL)</td>
			<td>A. Hartenstein</td>
			<td>PP05</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (plastic; diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>821473</td>
			<td>for zebrafish embryos</td>
		</tr>
		<tr>
			<td>pipette tips</td>
			<td>Sarstedt</td>
			<td>Blue (1000 &#181;l): 70762; Yellow (200 &#181;l): 70760002; White (10 &#181;L): 701116</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cell culture dishes (plastic; diameter 3 cm)</td>
			<td>TPP Techno Plastic Products AG</td>
			<td>93040</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cell culture dishes (plastic; diameter 6 cm)</td>
			<td>Sarstedt</td>
			<td>72690550</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile Petri dishes (plastic; diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td>for brain dissection</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Roth</td>
			<td>0601.1</td>
			<td></td>
		</tr>
		<tr>
			<td>4 % paraformaldehyde in 1 x PBS</td>
			<td>Sigma-Aldrich</td>
			<td>16005</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium nitrate tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1396</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol p.a. 100%</td>
			<td>Sigma-Aldrich</td>
			<td>46139</td>
			<td></td>
		</tr>
		<tr>
			<td>goat &#945;-mouse IgG (Fc specific) FITC conjugated</td>
			<td>Thermo Fisher Scientific</td>
			<td>31547</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Roth</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>high vacuum grease</td>
			<td>DOW CORNING</td>
			<td>3826-50</td>
			<td>silicon grease used for self-made glass bottom dishes</td>
		</tr>
		<tr>
			<td>magnesium sulfate heptahydrate</td>
			<td>Merck</td>
			<td>105886</td>
			<td></td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>Serva</td>
			<td>29198.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Tubulin, Acetylated antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua-Poly/Mount (mounting medium)</td>
			<td>Polyscience</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Biochrom</td>
			<td>L 7240</td>
			<td></td>
		</tr>
		<tr>
			<td>potasssion chloride</td>
			<td>Merck</td>
			<td>104938</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>Roth</td>
			<td>68514-61-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-X Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7471</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Synonym: Ethyl 3-aminobenzoate methanesulfonate</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioRad</td>
			<td>1610407</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco by Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase (Type 2)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17101015</td>
			<td>dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>pronase (from Streptomyces griseus)</td>
			<td>Roche</td>
			<td>11459643001</td>
			<td>distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Medium and solutions for cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 x PBS (Dulbecco&#39;s Phosphate Buffered Saline)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-169</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>CO2-independent medium</td>
			<td>Gibco by Life Technologies</td>
			<td>18045054</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>filtrated bovine serum (FBS)</td>
			<td>PAN-Biotech</td>
			<td></td>
			<td>individual batch</td>
		</tr>
		<tr>
			<td>glutamine 100 x</td>
			<td>Gibco by Life Technologies</td>
			<td>25030081</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium</td>
			<td>Gibco by Life Technologies</td>
			<td>11415049</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>PenStrep (10,000 units/mL)</td>
			<td>Gibco by Life Technologies</td>
			<td>15140148</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
	</tbody>
</table>

culture and transfection of zebrafish primary cells

Zebrafish 태아 투명 하 고 그대로 고 발전 등뼈 동물에서 동적 생물 학적 과정의 우수한 vivo에서 이미징 되므로 어머니 밖에 서 급속 하 게 개발 합니다. 그러나, 명료한 세포 유형 및 subcellular 구조의 형태학의 상세한 영상 전체 마운트에 제한 됩니다. 따라서, 우리 zebrafish 태아와 성인 조직 문화 라이브 1 차 셀을 효율적이 고 사용 하기 쉬운 프로토콜 설립.
간단 하 게, 2 dpf zebrafish 태아는 dechorionated, deyolked, 살 균, 그리고 콜라와 함께 단일 셀에 해리. 여과 단계 후 1 차 셀 유리 하단 요리에 도금 하 고 몇 일 동안 경작. 신선한 문화로 긴 기간 differenciated, 높은 해상도 confocal 이미징 연구에 사용할 수 있습니다. 문화 전기 myocytes와 폴 리-L-리 신 코팅에 저명한 되 고 신경 다른 셀 형식을 포함 합니다. 특히 라벨 subcellular 구조에 형광 마커 단백질에 의해, 우리는 또한 electroporation 프로토콜 뉴런을 포함 하 여 다른 세포 유형으로 플라스 미드 DNA의 transfection 수 있는 설립. 따라서, 연산자의 정의 된 자극, 복잡 한 셀 동작, 그리고 기본 zebrafish 세포의 세포내 역학 수 부과 높은 공간과 시간 해상도. 또한, 성인 zebrafish 두뇌를 사용 하 여 기본 자란 조건 뿐 아니라 설명된 분리 기술, 또한 성인 zebrafish 조직에 대 한 작업 설명 합니다.

그림 1G紋 myocytes 및 신경 같은 입자로 가장 풍부한의 클러스터 야생 타입 배아에서 파생 된 일반적인 문화의 전송된 빛 이미지를 보여줍니다. 더 쉽게 특정 세포 유형 식별, 유전자 변형 라인 형광 단백질의 세포 유형 특정 식이 될 수 있습니다 (그림 1 H)을 사용.
PCS2 +의 transfection-플?...

Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Culture and Transfection of Zebrafish Primary Cells

선물이 zebrafish 태아 transfection 라이브 셀 이미징 뿐만 아니라 성인 zebrafish 두뇌에서 1 차 셀을 준비 하는 프로토콜의 기본 셀 문화를 준비 하기 위한 효율적이 고 사용 하기 쉬운 프로토콜.

문화와 Zebrafish 기본 세포의 Transfection

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

culture-and-transfection-of-zebrafish-primary-cells

Research

JoVE Journal

Developmental Biology

12.3K 조회수.  Braunschweig University of Technology. 선물이 zebrafish 태아 transfection 라이브 셀 이미징 뿐만 아니라 성인 zebrafish 두뇌에서 1 차 셀을 준비 하는 프로토콜의 기본 셀 문화를 준비 하기 위한 효율적이 고 사용 하기 쉬운 프로토콜.

비디오: Culture and Transfection of Zebrafish Primary Cells

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

성인 Zebrafish의 뇌 유래 neurosphere를의 분리 및 문화

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

연구

발생학

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

일본 메추라기 배아의 노 른 골목 막에서 차 내배엽 상피 세포 배양

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

분리 및 Zebrafish의 배아에서 단일 세포의 특성

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

성인 및 노인 인간의 골격 근육에서 근육 조직 전구체 세포 / 차 근육 아세포의 준비 및 문화

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

수평 한 전체적인 거치 : 피부의 두껍고, 3 차원 조직 단면도를위한 비발 한 가공 그리고 화상 진찰 의정서

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

생활 그대로 Zebrafish에 단일 셀 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

분리와 인간 성인 그리고 태아 심장 표본에서 파생 하는 기본 Epicardial 세포의 문화

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

꼬마 선 충 생식 핵의 분포, 단백질, 및 골격을 전산 분석

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

유도 및 기본 인간 세포에서 세포 노화의 유효성 검사

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...