这项工作由索菲亚基金会之友（SSWO WAR-63）资助。

所有斑马鱼的饲养和实验都是根据伊拉斯谟MC和动物福利立法的制度准则进行的。受精后 5 天使用斑马鱼幼虫属于不需要正式伦理批准的实验类别，如荷兰法规所述。
1.获得受精后5天（dpf）野生型和tg（phox2bb：GFP）幼虫
<ol><li>设置野生型斑马鱼的繁殖，并在 15 cm 培养皿中收集 50 个卵在 HEPES 缓冲的 E3 培养基（以下简称 E3）中。使用这些鱼作为荧光激活细胞分选（FAC）的阴性对照。</li>
	<li>设置tg（phox2bb：GFP）斑马鱼的繁殖，并在15厘米培养皿中收集E3中的约300个卵。</li>
	<li>在28.5°C的培养箱中，以14小时/ 10小时的光照/黑暗循环将受精卵保持在E3（最多50个鸡蛋/培养皿）中。</li>
	<li>在 1 dpf 下，取出未受精的卵子，让受精的卵子发育到 5 dpf。</li>
	<li>选择 1 dpf 的 tg（phox2bb：GFP） 幼虫。</li>
</ol>2.野生型全幼虫解离
<ol><li>用0.016%Tricaine麻醉5 dpf幼虫。</li>
	<li>在含有 1 mL 10x 胰蛋白酶-EDTA 溶液的培养皿中使用剃须刀片将 30 条斑马鱼切成小块。</li>
	<li>将切碎的斑马鱼转移到总体积为 2 mL 的 10x 胰蛋白酶-EDTA 溶液的微量离心管中，并在冰上放置 3 小时。每小时用P1000移液器上下移液一次，以刺激解离。</li>
</ol>3.肠道隔离
<ol><li>在解剖显微镜下，将用 0.016% Tricaine 麻醉的 6-10 个 5 dpf 幼虫连续放置在 1.8% 琼脂糖平板（0.45 g 琼脂糖在 25 mL E3 中）上</li>
	<li>在斑马鱼的头上放一根昆虫针。</li>
	<li>用纸巾去除所有剩余的 E3。</li>
	<li>使用另一个昆虫别针隔离肠道，不要干扰任何其他器官。确保去除了蛋黄（补充图S1A）。</li>
	<li>分离后，检查肠道并根据需要去除所有非肠道物质（例如皮肤、脂肪、肝脏）。</li>
	<li>用镊子收集肠道并将其置于含有磷酸盐缓冲盐水（PBS）和10%胎牛血清（FCS）的微量离心管中，置于冰上。 注意：应分离至少 244 个肠子，将总解剖时间保持在 3 小时。这在两个人并行工作的情况下是可行的。</li>
</ol>4.肠道解离
<ol><li>解剖所有肠后，立即将微量离心管全速（13,800× g）离心30秒。</li>
	<li>取出PBS / 10%FCS，但保留少量（~100μL）以防止肠道变干。在含有 CaCl2 和 MgCl 2 的 HBSS 中加入 500 μL 2.17 mg/mL 木瓜蛋白酶以 解离细胞。</li>
	<li>用 2.5 μL 半胱氨酸 （1 M） 激活木瓜蛋白酶。</li>
	<li>将内脏在37°C的水浴中孵育10分钟。中途上下移液（5分钟后）以刺激酶组织消化。</li>
	<li>使用 35 μm 细胞过滤器将细胞转移到 FACS 管中，预润湿 0.5 mL PBS/10% FCS。</li>
	<li>以 0.5 mL 为步长，加入总共 2 mL 的 PBS/10% FCS，清洗过滤器。</li>
	<li>在4°C下以700× g 离心5分钟。</li>
	<li>除去上清液，将沉淀重悬于300μL PBS / 10%FCS中。</li>
	<li>加入 1 μg/mL 的 4'，6-二脒基-2-苯基吲哚 （DAPI） 以标记死细胞 （1：1,000） 并孵育 5 分钟以使其在 FACS 期间排除。</li>
</ol>5. 流式细胞仪富集
<ol><li>通过在流式细胞仪上记录 3,000个细胞 ，使用野生型样品设置分选细胞群的门（补充图S1B-E）。</li>
	<li>使用 100 μm 喷嘴，设置纯度的分选精度，并将含有 200 μL PBS/5% FCS 的收集管放入 FACS 机器中。</li>
	<li>通过排除双联体（图2B）和死细胞（图2C），将肠道的细胞悬液加载并将活的（DAPI阴性）单细胞分类到收集管中（图2C）。 注：对于tg（phox2bb）斑马鱼，仅检查GFP + 细胞的比例作为参考，因为所有单个活细胞都经过分选。</li>
	<li>细胞分选后将收集管保持在冰上。</li>
	<li>用血细胞计数器计数细胞悬液，包括用台盼蓝检查活力。如果存活率至少为 80%，请继续下一步。</li>
	<li>细胞现在可以处理 scRNAseq（即 10x Genomics Chromium 平台）。每个样品使用大约 20,000 个细胞。</li>
</ol>

处理斑马鱼幼虫的肠道细胞以进行单细胞 RNA 测序

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作者没有利益冲突需要披露。

在这里，我们提出了一种使用FACS分离和解离5 dpf斑马鱼幼虫肠道的方法。通过这种方法，使用10x Genomics Chromium平台，通过scRNA-seq成功收集和分析了不同的肠道细胞类型。我们选择了tg（phox2bb：GFP）斑马鱼系，因为我们想要一个迹象，表明活的ENS细胞也将被分离出来（图2D）。然而，重要的是要注意，这种方法可以很容易地扩展到其他感兴趣的斑马鱼品系，因为我们只?...

胃肠道 （GI） 是一个复杂的系统，在整体健康和福祉中起着至关重要的作用。它负责营养物质的消化和吸收，以及废物的消除 1,2。胃肠道由多种细胞类型组成，包括上皮细胞、平滑肌细胞、免疫细胞和肠神经系统 （ENS），它们紧密地相互通信以调节和维持适当的肠道功能 3,4,5。胃肠道发育的缺陷会对营养吸收、微生物群组成、肠脑轴和 ENS 等各个方面产生深远的影响，导致多种肠道神经肌肉疾病，例如先天性巨结肠和慢性假性肠梗阻 6,7。这些疾病的特征是由各种关键细胞（例如 Cajal 的间质细胞、平滑肌细胞和 ENS 6,8,9）的改变引起的严重肠道运动障碍。然而，胃肠道发育和功能障碍的分子机制仍然知之甚少。
斑马鱼是研究胃肠道发育和功能障碍的有价值的模式生物，因为它具有快速的胚胎发育、胚胎和幼虫阶段的透明度以及遗传可处理性 10,11,12,13,14。有许多表达荧光蛋白的转基因斑马鱼品系可供选择。这种品系的一个例子是tg（phox2bb：GFP）斑马鱼，通常用于研究ENS，因为所有phox2bb+细胞，包括肠神经元，都被标记为15,16。在这里，使用tg（phox2bb：GFP）斑马鱼系，我们提出了一种在受精后5天（dpf）幼虫肠道分离的方法，用于单细胞RNA测序（scRNA-seq）分析（图1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Trypsin (0.5%)-EDTA (0.2%)</td>
			<td>Sigma</td>
			<td>59418C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round bottom tube with cell-strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Round-Bottom Tube 5 mL (FACS tubes) snap cap</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Ranger v3.0.2</td>
			<td>10X Genomics</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#D-9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Olympus SZX16</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria III sorter machine</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with CaCl2 and MgCl2</td>
			<td>Gibco</td>
			<td>14025050</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>MS-222, Tricaine</td>
			<td>Supelco</td>
			<td>A5040-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma</td>
			<td>P4762</td>
			<td></td>
		</tr>
		<tr>
			<td>Seurat v3</td>
			<td>Stuart et&#160;al. (2019)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue&#160;</td>
			<td>Sigma&#160;</td>
			<td>Cat#T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

gut isolation from zebrafish larvae for single-cell rna sequencing

胃肠道 （GI） 执行一系列对生命至关重要的功能。影响其发育的先天性缺陷可导致肠道神经肌肉疾病，这凸显了了解胃肠道发育和功能障碍的分子机制的重要性。在这项研究中，我们提出了一种在受精后 5 天从斑马鱼幼虫中分离肠道的方法，以获得可用于单细胞 RNA 测序 （scRNA-seq） 分析的活细胞。该协议基于斑马鱼肠道的手动解剖，然后与木瓜蛋白酶酶解离。随后，将细胞提交荧光激活细胞分选，并收集活细胞进行scRNA-seq。通过这种方法，我们能够成功地识别不同的肠道细胞类型，包括上皮细胞、基质细胞、血液细胞、肌肉细胞和免疫细胞，以及肠道神经元和神经胶质细胞。因此，我们认为它是使用斑马鱼研究胃肠道在健康和疾病中的组成方面的宝贵资源。

The gastrointestinal (GI) tract is a complex system that plays a vital role in overall health and well-being. It is responsible for the digestion and absorption of nutrients, as well as the elimination of waste products1,2. The GI tract is composed of multiple cell types, including epithelial cells, smooth muscle cells, immune cells, and the enteric nervous system (ENS), which communicate closely together to regulate and maintain proper intestinal function3,4,5. Defects in the development of the GI tract can have far-reaching effects on various aspects such as nutrient absorption, microbiota composition, the gut-brain axis, and the ENS, leading to several enteric neuromuscular disorders, such as Hirschsprung disease and Chronic intestinal pseudo-obstruction6,7. These disorders are characterized by severe gut dysmotility caused by alterations in various key cells, such as the interstitial cells of Cajal, smooth muscle cells, and the ENS6,8,9. However, the molecular mechanisms underlying GI development and dysfunction are still poorly understood.
The zebrafish is a valuable model organism for studying GI development and dysfunction due to its rapid embryonic development, transparency during embryonic and larval stages, and genetic tractability10,11,12,13,14. Numerous transgenic zebrafish lines expressing fluorescent proteins are available. An example of such a line is the tg(phox2bb:GFP) zebrafish, commonly used to study the ENS, as all phox2bb+ cells, including enteric neurons, are labeled15,16. Here, using the tg(phox2bb:GFP) zebrafish line, we present a method for intestinal isolation of 5 days post fertilization (dpf) larvae for single-cell RNA sequencing (scRNA-seq) analysis (Figure 1).

作者聚焦：分离和分析斑马鱼幼虫的肠道细胞以研究胃肠道发育的转录组学方面 

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

Our research aims to investigate the transcriptomic changes occurring during gastrointestinal development and dysfunction in zebrafish larvae. Here, we provide a new protocol that allows isolation of different cell types within the zebrafish intestine, which has potential applications in understanding gastrointestinal health and disease. In recent years, there has been growing interest in exploring the interaction between the enteric nervous system and other intestinal cell types during development and disease.Understanding these complex dynamics using advanced techniques like single-cell RNA sequencing, holds the potential to reveal critical insights into enteric neuromyopathies, leading to more precise diagnostics and treatments. Using this protocol, we successfully identified various intestinal cell types, including epithelial, stromal, bloods, muscle, immune cells, and even enteric neurons and glia, which have not been captured before at this amount using similar approaches. Thus, we provide a reliable technique to study the gastrointestinal composition during development and dysfunction.

通过该协议，我们成功地从5个dpf幼虫中分离和解离了整个肠道。使用木瓜蛋白酶作为解离酶，我们显着增强了细胞活力，能够在 244 个分离的肠道中捕获 46,139 个涉及单个活细胞（占所有细胞的 6.4%）的事件（图 2A）。使用野生型全幼虫作为对照，以确保分选过程得到优化，从而实现有效的细胞鉴定和分选。整个幼虫可以用于我们只对活的单细胞进行分类（补充图S1B-E</...

Watch this Scientific Journal Video about Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development at JoVE.com

在这里，我们描述了一种在受精后 5 天从斑马鱼幼虫中分离肠道的方法，用于单细胞 RNA 测序分析。

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

我们的研究旨在调查斑马鱼幼虫胃肠道发育和功能障碍过程中发生的转录组学变化。在这里，我们提供了一种新的方案，允许在斑马鱼肠道内分离不同的细胞类型，这在了解胃肠道健康和疾病方面具有潜在的应用。近年来，人们越来越有兴趣探索肠道神经系统与其他肠道细胞类型在发育和疾病过程中的相互作用。使用单细胞RNA测序等先进技术了解这些复杂的动态，有可能揭示对肠道神经肌病的关键见解，从而实现更精确的诊断和治疗。使用该协议，我们成功地鉴定了各种肠道细胞类型，包括上皮细胞、基质细胞、血液、肌肉、免疫细胞，甚至肠神经元和神经胶质细胞，这些细胞以前从未使用过类似的方法捕获过。因此，我们提供了一种可靠的技术来研究发育和功能障碍期间的胃肠道成分。

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JoVE Journal

Developmental Biology

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

952 次观看.  Erasmus University Medical Center - Sophia Children's Hospital. 我们的研究旨在调查斑马鱼幼虫胃肠道发育和功能障碍过程中发生的转录组学变化。在这里，我们提供了一种新的方案，允许在斑马鱼肠道内分离不同的细胞类型，这在了解胃肠道健康和疾病方面具有潜在的应用。近年来，人们越来越有兴趣探索肠道神经系统与其他肠道细胞类型在发育和疾病过程中的相互作用。使用单细胞RNA测序等先进技术了解这些复杂的动态，有可?...