Name: isolation and characterization of single cells from zebrafish embryos
Uploaded: 2016-03-12T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).

이 프로토콜은 배아를 생산하는 라이브 성인 지브라 피쉬의 사용을 필요로한다. 배아는 조직 수집을 위해 수확. 이 실험을 수행하기 위해 적절한 윤리 리뷰 보드의 승인을 얻기 위해 필수적이다. 1. 단계적 배아를 구합니다 <ol><li> 실험 전날, 번식을위한 건강한 성인 제브라 피쉬를 준비합니다. 한 남자와 사육 탱크에 명확한 구분선의 반대편에 한 여자를 놓습니다. </li><li> ?...

<ol>
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본원에 기재된 방법은 하나의 심장 유전자 집합의 발현을 평가하기 위해 마이크로 유체 보조 된 단일 셀 캡쳐 시스템에서 사용하기 위해 제브라 배아 심장 선조 세포 집단을 풍부하게하는 세포 형 특이 적 프로모터의 제어하에 형광 단백질의 발현을 사용하여 세포. FACS 레이저 여기 및 발광 기능은 원하는 형광 물질 (들)과 호환되는지 다만,이 방법은 형광 리포터 라인에 대해 사용될 수있다. 많은...

세포 및 분자 생물학의 현재 대부분의 연구는 인구의 평균을 기준으로합니다. 작은 인구가 생물학적 과정과 질병의 결과에 중요한 역할을 할 수 있기 때문에, 중요한 생물학적 이벤트는 이러한 기존의 인구 기반의 분석에 의해 마스크 될 수있다. 단일 세포 수준에서 이질적인 집단에서 유전자 발현을 이해하는 것은 (그리고있다) 관련 생물학적 및 임상 통찰력 1,2로 이어질 수 있습니다. 배아 발달 과정에 관심 셀의 큰 모집단에서 전구 세포는 종종 소수가있는 도전 결국 세포의 운명 결정 (3)을 개시 유전자 발현의 미묘한 변화를 감지 할 수있다. 마찬가지로, 단일 셀 타입 미세 4에 응답하여 상이한 발현 프로파일을 가질 수있다. 예를 들어, (예., 대동맥 또는 신장) 같은 기관 또는 다른 기관에 거주 내피 세포는 일반적인 morp을 공유에도 불구하고 상당한 이질성을 전시hological 및 기능 기능 5. 또한, 동일한 종양 채우는 암세포는 단일 세포 수준에서 6 분 프로필 또는 돌연변이를 변화 할 수있다. 모델 시스템에서, 하나의 세포에서 transcriptomics 성공적으로 새로운 세포 집단을 식별 세포 분화 동안 발생하는 중간 상태를 특징으로하고, 7, 8, 9를 자극에 차등 세포 반응을 공개했다. 이러한 통찰력은 기존의 인구 기반 연구에서 마스크 된 것이다. 제브라 피쉬 배아 줄기, 전구의 대단히 과소 활용 소스, 그리고 개발 과정에서 단일 세포의 이질성 및 세포 정체성의 분자 규제의 질문을 탐험 세포를 분화. 그들의 매우 틀에 박힌, 생체 개발 및 유전자 조작의 용이성은 그들이 방법 10,11위한 훌륭한 모델 시스템합니다. 특히, 단일 세포 세대의 해석에 큰 제한E 발현 데이터는 개발 중에 신규 중간체 셀 상태의 신뢰성 식별 조직 수집 9 조심 타이밍을 필요로한다는 것이다. 이것은 캡처 세포 이질성 연령 의존적 세포 분화 제시 유전자 발현 한 시점에서 조직 내의 얼룩이보다 이질성을 나타낸다는 것을 보장 할 필요가있다. 마우스에 비해, 제브라 피쉬 배아 발달 정확하게 배아 (12)의 다수를 통해 동기화 될 수있다. 또한, 큰 크기의 클러치, 제브라 피쉬 배아 줄기 세포 및 선조 세포의 풍부한 공급원으로서 사용될 수있다. 이 프로토콜은 제브라 피쉬 배아 세포를 분리하고 QRT-PCR 유전자 발현 분석을위한 시판 통합 마이크로 유체 회로 (IFC) 칩 autoprep 시스템을 사용하여 단일 세포를 포착하는 방법을 설명한다. 이 프로토콜은 전체 처리량을 포함하는 임의의 다중 검정법 빠르게 양도 될 수있다휴대 이질성 (13)의보다 포괄적 인 분석이 가능 사체 서열. 또한, 기존의 유전자 발현 분석에 여러 가지 이점을 제공한다. 단일 세포 분리 프로토콜 하류 어플리케이션에 포함되어 손상된 세포의 비율을 FACS 감소 후 높은 생존율을 산출한다. IFC를 사용하여 캡처 된 세포를 직접 포획 율을 평가하고 세포 형태 학적 상태를 평가하는 것으로 관찰 될 수있다. 또한,이 프로토콜은 미세 유체 세포 캡처 기술 만 표시 형질 전환 어류 라인과 접근을 필요로 제브라 피쉬 연구 커뮤니티에 광범위하게 적용 할 수있다. 원리 증명으로, 심장 전구 세포로부터 유도 된 단일 세포를 격리시키고, IFC 칩 캡처하고 심장 분화 마커의 상대적인 존재 량 QRT-PCR에 의해 측정 하였다. 단일 세포 수준에서의 유전자 발현 분석은 심장 전구 세포가 differe 공존 것을 증명ntiating 자손. 심장 전구 세포의 단일 셀 프로파일에서 얻은 통찰력은 기존의 인구 기반의 분석에 마스크되었을 수 있습니다 척추 개발하는 동안 심장 전구 세포 간의 유전자 발현 패턴의 이질성에 광명을 비춰 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="816">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplies</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">11254-20</td>
			<td>For removing the chorion from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube 2 mL</td>
			<td style="width:111px;">GeneMate</td>
			<td style="width:103px;">C-3261-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40 um cell strainer</td>
			<td style="width:111px;">Biobasic</td>
			<td style="width:103px;">SP104151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm culture dish</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">351008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACS tubes topped with 35 um cell strainer</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P20 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005091</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IFC chip manufacturer&#39;s protocol</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-6117</td>
			<td>Version 100-6117 E1 was used in representative experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wide bore pastuer glass pippette</td>
			<td style="width:111px;">VWR</td>
			<td style="width:103px;">14673-010</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult wild type zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used AB line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult transgenic zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used Tg(nkx2.5:ZsYellow)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents for cell dissociation</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double distilled water</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Sea Salt</td>
			<td style="width:111px;">Instant Ocean</td>
			<td style="width:103px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">S271-3</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">P5405</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">S6014</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leibovitz&#39;s L-15</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">21083-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">03-600-511</td>
			<td>Heat inactivate; any brand of FBS should be fine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 1 -TrypLE</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">12605-010</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 2 - FACSmax</td>
			<td style="width:111px;">Genlantis</td>
			<td style="width:103px;">T200100</td>
			<td>Store -20; thaw on ice; bring to room temp before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pronase (optional)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">P5147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L/D Dye - Sytox Blue</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">S34857</td>
			<td>Any live/dead stain suitable for flow cytometry will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents for IFC plate use and qRT-PCR</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene-specific probes</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>Probes will vary by experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe ef1a</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03432748_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe gata4</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03443262_g1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe nk2-5</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03074126_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe myl7</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03105700_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe vmhc</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03431136_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe isl1</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03425734_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4369016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents listed in IFC manufacturer&#39;s protocol</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 Reagent Kit</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5319</td>
			<td>Reagents for loading cells onto IFC plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ambion Single Cell-to-CTTM</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4458237</td>
			<td>Reagents for reverse transcription and pre-amplification steps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Molecular Biology Quality Water</td>
			<td style="width:111px;">Corning</td>
			<td style="width:103px;">46-000CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 IFC for PreAmp (5-10 um)</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5757</td>
			<td>IFC plate for small cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 AutoPrep machine</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5477</td>
			<td>For IFC plate use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">Z359629</td>
			<td>For counting cells and assessing cell size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For removing embryos from chorion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue culture microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For assessing single cell digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS machine</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For isolating cells of interest</td>
		</tr>
	</tbody>
</table>

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.

원리의 증거로, 유전자 발현이 심장 개발하는 동안 차별화 역학을 탐구하는 평가 하였다. 제브라 피쉬, 심장 조상들은 선형 심장 튜브를 형성하도록 융합 전방 측 방향 플레이트 중배엽 마이그레이션 중배엽 세포 집단에서 발생한다. 융합에 앞서, 심장 선조 심장 선조 16,17의 초기 특정 마커 생각된다 전사 인자 nkx2.5 (NK2의 호 메오 5) 표현하기 시작한다. 여?...

Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

Isolation and Characterization of Single Cells from Zebrafish Embryos

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 ...

The overall goal of this experiment is to isolate single cells from early stage zebrafish embryos for their capture in a microfluidic-based single-cell multiplex system to assess the gene expression of individual cells. This method can help answer key questions in the developmental biology field, such as what transcriptional changes do cells undergo during specification and differentiation. The main advantage of this technique is that it is an unbiased approach for revealing the heterogeneity that is masked in conventional population-based techniques.Generally, individuals new to this technique will struggle because generating the viable preparations of single cells is technically challenging but essential for downstream analysis. Once mastered, processing the whole embryo samples into single lysed cells on an integrated microfluidic chip can be completed in six hours if steps are performed properly. While attempting this procedure, it's important to remember to make sure that only single cells are captured on the microfluidic device.After breeding adult wild-type and transgenic zebrafish, collect embryos at 15-minute intervals according to facility and institution-approved standard operating procedures. At least two hours later, transfer 100 to 300 fertilized embryos from a single timepoint into a new Petri dish containing fresh egg water. And use fine forceps to manually remove the chorion from each embryo.Next, use a wide bore glass pipette to transfer the embryos into a 2-milliliter microcentrifuge tube in a minimum volume of egg water. When all of the embryos have been transferred, replace the water with 1 milliliter of ice cold egg water and submerge the tube in ice for 20 minutes. After the euthanization, use a wide bore glass pipette to remove the supernatant.And a P-1000 pipette to add fresh egg water to the embryos two times. After the second wash, replace the egg water with 1 milliliter of de-yolking buffer and use a P-1000 pipette to triturate the embryos eight to 12 times until the yolk is dissolved and only the bodies of the embryos are visible. Collect the tissue by centrifugation.Then, use the pipette to gently remove the supernatant without disturbing the tissue pellet, and re-suspend the cells in 1 milliliter of fresh egg water. After washing the embryos two more times, re-suspend the pellet in 1 milliliter of room temperature cell dissociation reagent-1. Then place the tube on its side for a 10-minute incubation at room temperature.With gentle trituration every two to three minutes to prevent clumping. At the end of the incubation, spin down the cells again and re-suspend the pellet in 1 milliliter of cell dissociation reagent-2. Incubate the cells horizontally at room temperature for another five to 15 minutes with gentle trituration every two to three minutes.Every five minutes, dilute 2 microliters of supernatant in 18 microliters of FACS buffer, and dispense the cells as a droplet onto a cell culture dish. Place a coverslip over the sample and use a tissue culture microscope to assess the digestion progression at 10 and 20x magnifications. When the preparation appears to be a mix of primarily single cells with some small and large cell clusters and a few mostly intact embryo bodies, spin down the cells and re-suspend the pellet in 1 milliliter of cold FACS buffer.Next, wet a 40-micron cell strainer with FACS buffer. And filter the cells through the strainer into a 35-millimeter cell culture dish. After spinning down the cells again, re-suspend the pellet in 100 microliters of FACS buffer, and count the number of viable cells by Trypan blue exclusion.Next, dilute the sample to 5 times 10 to the 6th cells per milliliter. And reserve 10 to 20%of the cells for the unstained control. Stain the rest of the cells with a fluorescent live/dead discrimination dye.Then wet a 35-micron cell strainer capped FACS tube with 20 microliters of FACS buffer, and add the cells to the strainer, collecting them by gravity. Using the illustrated gating strategy, sort the single live cells expressing the fluorescent marker. Verifying the gating with the double-labled cells.Sort 2, 000 to 4, 000 cells from the population of interest into 5 microliters of cold FACS buffer in a microcentrifuge tube on ice. Then transfer 1 microliter of the sorted cells into a new FACS tube with 100 microliters of FACS sorting buffer and a 1 to 1, 000 dilution of live/dead stain. And run the cell sample using the sorting gates to assess the post sort viability.Next, load 1 microliter of cells diluted in 9 microliters of FACS buffer onto a hemocytometer to determine the cell concentration and average diameter. Now select an integrated microfluidic circuit, or IFC chip, of the appropriate size for the average cell diameter of the sample, and prime the fluidics according to the manufacturer's instructions. Load the cells onto the plate according to the manufacturer's instructions, and load the plate into a compatible fluidic machine.Then run an appropriate cell loading script for the IFC chip to push the cells into the capture lanes. To confirm that the cells are lodged in the capture sites, mount the plate on a microscope equipped with a plate adapter and obtain brightfield and fluorescence images of each capture site at a 10x magnification. The cells can then be processed for downstream assessment of their target gene expression by QRT-PCR.At the 18-somite stage, fluorescence microscopy can be used to verify the ZsYellow expression of the embryos. After sorting, cells with a range of diameters are obtained. The IFC plate can be used to capture cells with a diameter of interest.For example, in this experiment, cells five to 10 micrometers in diameter. As expected, the sorted and captured cells from 18-hour post-fertilization embryos expressed the ZsYellow fluorescent marker. Here, QRT-PCR was used to assess the relative gene expression of specific genes of interest in 40 single cells from cell capture sites on an IFC plate.Since the range of cycle threshold values for the housekeeping gene was too broad for comparing gene expression between samples and many cycle threshold values exceeded 30, The values were visualized as a heat map. Comparing across samples, a substantial heterogeneity in gene expression was observed in this experiment. With cells classified as Types 1 through 5 based on their gene expression pattern.Once mastered, processing the whole embryo samples into single lysed cells on an integrated microfluidic chip can be completed in six hours if the steps are performed properly. While attempting this procedure, it's important to remember that only single cells are captured on the microfluidic chip. Following this procedure, other methods like high-throughput single cell sequencing can be performed to fully characterize the transcriptomes of single cells.After its development, this technique paved the way for researchers in the field of developmental biology for exploring cellular heterogeneity in early developing embryos. After watching this video, you should have a good understanding of how to isolate single cells from zebrafish embryos, capture single cells in a microfluidic base single-cell multiplex system and assess the gene expression of individual cells.

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

인간의 골격근에서 기본 근육 조직 세포와 섬유 아 세포의 분리 및 정량 면역 세포 화학 특성

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

연구

발생학

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

쥐 머리 Branchiomeric 근육에서 분리 및 위성 세포의 특성

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Zebrafish의 배아에서 내인성 수용체의 친화력 레이블 검출

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Single-cell Photoconversion in Living Intact Zebrafish

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 ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

절연 및 인간의 탯 유래 중간 엽 줄기 세포 Preterm와 기간 유아에서의 특성화

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

골격 근 생검 절차에서 인간 근육 선조 세포의 격리, 배양, 특성화 및 분화

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

초기 마우스 배아에서 시퀀싱을 위한 작은 RNA 라이브러리 의 준비

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

제브라피시 배아에서 나이브 Blastoderm 의 세대

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

마우스와 인간의 치아에서 단일 세포의 신속한 격리

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...