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).

该协议要求使用的活，成年斑马鱼产生的胚胎。将胚收获组织收集。重要的是要获得相应的伦理审查委员会批准进行这项实验。 1.获取胚胎上演<ol><li>在实验前一天，繁殖准备健康，成年斑马鱼。放置一个男性和一个养殖池一个明确的分隔两侧一名女性。 </li><li>重复1.1所必需的足够胚胎生产下游应用尽可能多的养殖池。获得从野生型鱼和转基因鱼胚胎表达的感兴趣的细胞类...

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本文所描述的方法使用荧光蛋白的表达下的细胞类型特异性启动子的控制，以丰富从斑马鱼胚胎中的微流体辅助单细胞捕获系统中使用的心脏祖细胞群，以评估在单个心动基因的一个子集的表达细胞。只要FACS激光激发和发射能力与所选择的荧光团（多个）相容，可以使用任何荧光报道线路此方法。许多斑马鱼记者线已经存在，并且可以在少至3个月后将产生携带新颖记者转基因鱼。此外，此工作?...

细胞和分子生物学目前大多数研究是基于人群的平均水平。然而，重要的生物事件可能由这些传统的以人群为基础的分析被屏蔽，因为较小的群体可以在生物过程和疾病预后扮演着重要的角色。理解在不同的人群的基因表达在单细胞水平可以（有）导致相关的生物和临床见解1,2。关注胚胎发育研究，在细胞的人口较多，祖细胞往往代表性不足，使得它具有挑战性的检测基因表达微妙的变化，最终引发细胞命运决定3。同样地，一个单一的细胞类型可响应于微环境4具有不同的表达谱。例如，在相同的器官或在不同的器官（ 例如 ，主动脉或肾）驻地内皮细胞表现显著异质尽管共享共同morphological和功能特点5。此外，癌细胞填充相同的肿瘤还可以在单细胞水平6不同分子型材或突变。 在模型系统中，单个细胞转录组已成功确定了新的细胞群，其特征细胞分化过程中发生的中间状态，并透露差细胞对刺激7,8,9。这样的见解会被掩盖了传统的基于人口的研究。斑马鱼的胚胎是干，祖的极大充分利用的源代码，并在开发过程中探索单细胞的异质性和细胞身份的分子调控问题分化细胞。他们的高度千篇一律， 体外发育和易于遗传操作使其成为这种方法10,11极好的模型系统。具体而言，主要的限制单细胞根的解释Ë表达数据是在开发过程中新的中间细胞状态的可靠的鉴定需要组织收集9非常小心时机。这是必要的，以确保捕获的细胞之间的异质性表示在单个时间点的组织内的异质性，而不是异质由年龄依赖性细胞分化呈现的基因表达。相比小鼠，斑马鱼胚胎发育可以精确在大量的胚胎12的同步。此外，具有大的离合器的尺寸，斑马鱼的胚胎可以用作干细胞和祖细胞的丰富来源。 这个协议描述来隔离从斑马鱼胚胎细胞和使用用于QRT-PCR的基因表达分析的市售集成微流体回路（IFC）芯片和Autoprep设备系统捕获单细胞的方法。该协议可迅速移植到任何高通量测定复包括全转录组测序，使细胞的异质性13的更全面的分析。它还提供了多种优势，传统的基因表达分析。单细胞分离协议产生的FACS后高存活率，这降低了包含在下游应用中受损细胞的比例。通过使用IFC，捕获细胞可以直接观察来评估捕获速率和形态学评估细胞的健康。此外，该协议是广泛适用于斑马鱼研究界，只需要一个标有转基因鱼线，并获得微流体细胞捕获技术。 作为一个原则的证明，从心脏祖细胞衍生的单细胞分离和IFC芯片上捕获，然后心脏分化标志物的相对丰度被定量RT-PCR检测。在单细胞水平的基因表达分析表明，心脏祖细胞与各色共存ntiating后代。从心脏祖细胞的单细胞分析的洞察力可以在脊椎动物的发育过程中心脏祖细胞中的基因表达模式的异质性，这可能在传统的基于人群的分析被掩盖线索。

<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.

作为一个原则的证明，基因表达进行了评估心脏发育过程中，探索差异化的动态。在斑马鱼，心脏祖细胞从迁移到前侧板中胚层，他们融合形成线性心脏管细胞的中胚层人口出现。之前的融合，心脏祖细胞开始表达转录因子NKX2.5（NK 2同源5），它被认为是心脏祖16,17的最早特异性标记。在这里，先前描述的BAC转基因鱼的Tg（NKX2.5：ZsYellow）18，</su...

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.

分离和斑马鱼胚胎单细胞的表征

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

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

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

科研

发育生物学

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

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

从斑马鱼胚胎内源性受体亲和标记检测

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

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

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

活完好斑马鱼的单细胞 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

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

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

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

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

为早期小鼠胚胎测序准备小型RNA库

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

从斑马鱼胚胎中产生幼稚的胚泡外植体

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

从小鼠和人类牙齿中快速分离单个细胞

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