这项工作得到了中国自然科学基金（82070450 to C.T.和 82170466 to L.Z.）和中国博士后科学基金（7121102223 to F.L.）的资助。

以下所有动物程序均由苏州大学机构动物护理和使用委员会批准。
1.试剂和材料准备
<ol><li>利用不含钙和镁的 1x PBS 和 2.5 U/mL 肝素钠盐制备灌注溶液。储存在4°C，使用时在冰上预冷。</li>
	<li>用HBSS稀释1250CDU/mL胶原酶II和3000U/mL DNase I，制备含有125 CDU/mL胶原酶II和60U/mL DNase I的解离试剂A。储存在4°C直至使用。</li>
	<li>通过将 1 mL 不含 EDTA 的胰蛋白酶（不含酚红）稀释到 1 mL 的 1x PBS 中，制备含有终浓度为 0.12% 胰蛋白酶的解离试剂 B。储存在4°C直至使用。</li>
	<li>在 1x PBS 中制备 10 mL 的 1.5% FBS 和 10 mL 的 5% FBS。使用1.5%FBS汇集颈动脉，并在使用前保持冰敷。使用5%FBS中和消化并在室温（RT）下储存。</li>
	<li>获取实验材料，包括 1 mL 注射器、20 mL 注射器和 26-G 针头、六孔细胞培养板、1.5 mL 离心管、40 μm 无菌细胞过滤器和冰容器。</li>
</ol>2、设备准备
<ol><li>对所有手术设备进行消毒，包括立体显微镜、解剖剪刀、显微解剖剪刀和细尖镊子。</li>
	<li>打开自动细胞计数仪并将其保持在室温。</li>
	<li>提前将水浴打开至37°C。</li>
	<li>使用前将微量离心机预冷至4°C。</li>
</ol>3.小鼠颈动脉的隔离
<ol><li>通过腹膜内注射（每10g体重0.24mL）使用1.25%三溴乙醇麻醉小鼠，并在3分钟后翻转鼠标以检查是否有矫正反射。然后，通过颈椎脱位对小鼠实施安乐死，并将其仰卧。用75%的酒精喷洒小鼠的皮肤。</li>
	<li>使用消毒的解剖剪刀打开皮肤并露出胸腔。切开隔膜。</li>
	<li>继续向上切割至下颌，小心地去除多余的结缔组织和脂肪，露出颈动脉。</li>
	<li>切开小鼠的下腔静脉，使血液从闭合循环中流出。</li>
	<li>用注射器缓慢连续地将 20 mL 预冷灌注溶液注入左心室。 注意：如果灌注成功，肝脏、脾脏和肾脏等富含血液的器官将变成灰白色。</li>
	<li>在立体显微镜下用显微解剖剪刀剥离颈动脉周围的所有脂肪和结缔组织。 注意：此步骤应缓慢而小心地进行，以防止颈动脉受损。</li>
	<li>从小鼠中分离颈动脉，用1x PBS洗涤以再次冲洗血液，并在冰上转移到含有1.5%FBS的六孔板中。</li>
</ol>4.将颈动脉消化成单细胞悬液
<ol><li>使用显微解剖剪刀纵向解剖颈动脉，并平放在另一个含有 1 mL 1.5% FBS 的 6 孔细胞培养板中。</li>
	<li>用 1.5% FBS 小心冲洗内膜以去除残留的血液。 注意：冲洗应轻柔缓慢，以免损伤内皮细胞。</li>
	<li>使用显微解剖剪刀在1.5%FBS中将每条颈动脉切成约2mm2 组织块。</li>
	<li>将这些组织块转移到具有1mL宽孔尖端的1.5mL离心管中，在4°C下以400× g 离心5分钟以将组织沉入底部。</li>
	<li>弃去上清液并将组织重悬于500μL解离试剂A中。</li>
	<li>将试管置于37°C水浴中1小时。每 10 分钟用 1 mL 移液管轻轻移液。</li>
	<li>向试管中加入 500 μL 5% FBS，并用 1 mL 移液管充分混合。</li>
	<li>将细胞悬液通过40μm无菌细胞过滤器过滤到新的1.5mL离心管中，并在4°C下以400× g 离心5分钟。</li>
	<li>小心地取出上清液，将细胞沉淀重悬于200μL解离试剂B中。</li>
	<li>置于37°C水浴中，再消化5分钟。在消化过程中每 2 分钟轻轻移液一次，以完全解离成单细胞悬浮液。</li>
	<li>使用200μL的5%FBS终止消化反应，并在4°C下以400× g 离心5分钟。</li>
	<li>弃去上清液，将细胞沉淀重悬于冰上的100μL1x PBS中。</li>
</ol>5. 细胞悬液检查和单细胞RNA测序
<ol><li>将 10 μL AO/PI 溶液加入 10 μL 单细胞悬浮液中，并用 20 μL 移液管轻轻混合。</li>
	<li>将 20 μL 混合物移入腔室载玻片中并等待 1 分钟。</li>
	<li>将载玻片装入自动细胞计数仪中。</li>
	<li>选择 AO/PI 活力 检测，然后等待质量报告。</li>
	<li>使用单细胞 3ʹ 试剂盒在单细胞控制器上的乳液中生成单细胞凝胶微球，并按照制造商的方案在热循环仪中扩增带条形码的 cDNA。 注：这里使用的是 Chromium Single Cell 3ʹReagent Kits v3。</li>
	<li>在具有双端 150 bp （PE150） 读数策略的测序仪上进行测序。使用软件应用程序 （Cell Ranger） 使用 mkfastq 管道将原始碱基调用文件转换为 fastq 文件。然后，使用 Cell Ranger 的 Count Pipeline 进行比对、过滤、条形码计数和唯一分子标识符 （UMI） 计数，以生成特征条形码矩阵13。</li>
	<li>使用 R 包 Seurat14 执行数据降维和可视化。<ol><li>首先，Seurat 的 Read10X（） 函数读取 Cell Ranger 管道的输出，然后使用计数矩阵创建 Seurat 对象。然后，通过修拉 的subset（） 函数过滤出表达<200或>4000个基因或线粒体基因比例超过10%的细胞。</li>
		<li>使用 NormalizeData （） 和 FindVariableFeatures（） 分别对数据进行归一化并识别 2000 个高度可变的特征。</li>
		<li>对缩放数据执行主成分分析 （PCA），并使用 dims = 30 和分辨率 = 0.5 对像元进行聚类。最后，使用 RunUMAP （） 函数可视化单细胞数据集，使用 FindAllMarkers（） 查找每个簇生物标志物。</li>
	</ol></li>
</ol>

小鼠颈动脉的两步细胞消化

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作者没有要披露的利益冲突。

在这里，我们提供了从野生型小鼠颈动脉制备高质量单细胞悬浮液的详细方案，其中构建了一种整合胶原酶/ DNase和胰蛋白酶消化过程的两步消化方法。经过对单细胞悬液的质量检查，我们发现它满足了单细胞测序的要求，细胞活力超过85%，细胞浓度高。此外，经过单细胞数据处理，成功检测到颈动脉中的多种细胞类型，包括VSMCs、成纤维细胞、ECs和Mφ/DCs。
目前制备颈动脉单?...

动脉粥样硬化是一种慢性炎症性疾病，与高血压、高脂血症和血流动力学等危险因素有关1。颈动脉分叉容易发生血流动力学改变，导致颈动脉斑块形成。颈动脉粥样硬化的临床表现可以是急性的，如卒中和短暂性脑缺血，也可以是慢性的，如复发性短暂性脑缺血和血管性痴呆2。从机械上讲，颈动脉斑块是病理条件下不同血管壁细胞与各种血细胞相互作用的结果。因此，揭示生理和病理条件下的颈动脉血管单细胞图谱对于预防和治疗颈动脉斑块的发展尤为重要。
单细胞 RNA 测序是生物学研究中最强大的技术之一，因为它具有超高分辨率和检测来自相同细胞类型生物体的细胞异质性 3,4。研究人员使用单细胞RNA测序在许多领域进行研究，例如心血管疾病5和癌症6。然而，快速准确地将组织分离成单细胞仍然是主要挑战之一。酶解离是一种常用的方法，主要包括胶原酶、木瓜蛋白酶、胰蛋白酶、DNase 和透明质酸酶。具体来说，胶原酶是单细胞消化的主要酶种，主要水解结缔组织中的胶原成分。不同的胶原酶类型适用于不同组织的解离，如乳腺7、肾小球8、虹膜角角9、膝关节10、主动脉11、肺12。由于不同组织独特的生理特性，使用相同的方法解离可能会在获取单个细胞时造成许多麻烦，例如细胞活力低、细胞数量少、细胞碎片大等。因此，针对不同组织的消化方法的发明对于制备高质量的单细胞悬浮液至关重要。
该方案旨在开发一种两步细胞消化方法，以制备野生型小鼠颈动脉的单细胞悬浮液。根据颈动脉的特点，我们将胶原酶/DNase与胰蛋白酶联合使用，以获得高质量的小鼠颈动脉单细胞悬液，因为胶原酶可以水解颈动脉组织的胶原蛋白，颈动脉组织被胰蛋白酶进一步消化成单细胞悬浮液。采用吖啶橙/碘化丙啶（AO/PI）双荧光计数检测细胞活力和浓度，发现单细胞悬液满足单细胞测序要求，细胞活力超过85%，细胞浓度高。经过单细胞数据处理，在消化后的正常小鼠颈动脉中鉴定出血管平滑肌细胞（VSMCs）、成纤维细胞、内皮细胞（ECs）和巨噬细胞和树突状细胞（Mφ/DCs）等4种细胞类型。该协议的优点是：1）可以识别颈动脉中的多种细胞类型，2）细胞活力保存良好，3）无需特殊设备即可轻松重复。该协议适用于有兴趣研究小鼠颈动脉单细胞多组学的研究人员。该方案还可能有助于通过适当的修改解离其他血管。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% EDTA-free trypsin</td>
			<td>Beyotime</td>
			<td>C0205</td>
			<td>Dilute 1 mL of 0.25% EDTA-free trypsin into 1 mL of 1x PBS.</td>
		</tr>
		<tr>
			<td>0.9% NaCl saline solution</td>
			<td>Beyotime</td>
			<td>ST341</td>
			<td>Dilute the heparin sodium solution into a final concentration of 10 mg/mL</td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>&#160;SKJYLEAN</td>
			<td>sk-r009</td>
			<td>To perform cardiac perfusion</td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>KIRGEN</td>
			<td>KG2211W</td>
			<td>To centrifuge the tissue piece and cell suspension</td>
		</tr>
		<tr>
			<td>20 mL syringes</td>
			<td>SKJYLEAN</td>
			<td>sk-r013</td>
			<td>To perform cardiac perfusion</td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>JETBIOFIL</td>
			<td>css010040</td>
			<td>To filter undigested tissue fragments</td>
		</tr>
		<tr>
			<td>AO/PI kit</td>
			<td>Hengrui Biological</td>
			<td>RE010212</td>
			<td>To identify whether the cell is alive or dead</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Countstar</td>
			<td>Mira FL</td>
			<td>To analyze the cell morphology and cell viability of digested carotid vascular cells</td>
		</tr>
		<tr>
			<td>Cell Ranger software</td>
			<td>10&#215; Genomics</td>
			<td>3.0.2</td>
			<td>To process Chromium single-cell RNA-seq output and perform clustering and gene expression analysis</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39;Reagent Kits v3</td>
			<td>10&#215; Genomics</td>
			<td>1000075</td>
			<td>To prepare single-cell RNA-seq libraries of single-cell suspension</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td>Dilute with HBSS to a final concentration of 125 CDU/mL</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington</td>
			<td>LS002140</td>
			<td>Dilute with HBSS to a final concentration of 60 U/mL</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>HyClone</td>
			<td>SH30088.03</td>
			<td>Termination of the digestion reaction</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution&#160;</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td>Store at the room temperture</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Solarbio Life Science</td>
			<td>H8060</td>
			<td>Dilute with 0.9% NaCl to a final concentration of 10 mg/mL</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002560</td>
			<td>Applied for spining down the tissues and cell pellets</td>
		</tr>
		<tr>
			<td>NovaSeq 6000&#160;</td>
			<td>Illumina</td>
			<td>N/A</td>
			<td>Sequencer</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Solarbio Life Science</td>
			<td>P1000</td>
			<td>Used for cardiac perfusion and resuspension of cells</td>
		</tr>
		<tr>
			<td>Seurat package- R</td>
			<td>Satija Lab</td>
			<td>3.1.2</td>
			<td>To performed dimensionality reduction, visualization, and analysis of scRNA-sequencing data</td>
		</tr>
		<tr>
			<td>Six-well cell culture plates</td>
			<td>NEST</td>
			<td>703002</td>
			<td>Place the vascular tissue</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Jinghong</td>
			<td>DK-S22</td>
			<td>Keep the digestion temperature at 37 &#176;C</td>
		</tr>
	</tbody>
</table>

preparation of a single-cell suspension from mouse carotid arteries for single-cell sequencing

颈动脉是颈部的主要血管，为大脑提供血液和氧气，但当颈动脉被斑块堵塞时，就会发生颈动脉狭窄。在单细胞水平上揭示颈动脉的细胞组成对于治疗颈动脉粥样硬化至关重要。然而，目前尚无现成的方案用于制备颈动脉单细胞悬浮液。为了获得在单细胞水平上解离正常颈动脉且对细胞损伤较小的合适方案，我们设计了一种两步消化方法，通过整合胶原酶/DNase 和胰蛋白酶的消化过程。采用吖啶橙/碘化丙啶（AO/PI）双荧光计数检测细胞活力和浓度，发现单细胞悬液满足单细胞测序要求，细胞活力超过85%，细胞浓度高。单细胞数据处理后，在每个颈动脉细胞中检测到每个细胞的中位数~2500个转录本。值得注意的是，可同时检测到正常颈动脉的多种细胞类型，包括血管平滑肌细胞 （VSMC）、成纤维细胞、内皮细胞 （EC） 以及巨噬细胞和树突状细胞 （Mφ/DC）。该方案可用于制备具有适当修饰的来自其他组织的血管的单细胞悬浮液。

该方案描述了一种用于制备小鼠颈动脉单细胞悬浮液的两步细胞消化方法（图1）。这种两步细胞消化方法将胶原酶/DNase与胰蛋白酶相结合，可有效解离小鼠颈动脉血管壁，获得用于单细胞测序的高质量单细胞悬液。解离后，通过自动细胞计数仪计算细胞浓度和细胞活力。明场显示消化后颈动脉血管细胞的形态，AOPI双荧光染色同时检测细胞活力（图2A，B</strong...

Watch this Scientific Journal Video about Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy at JoVE.com

作者聚焦：血管组织解离和探索用于靶向治疗的单细胞亚簇 

在这里，我们描述了一种两步细胞消化方案，用于制备小鼠颈动脉的单细胞悬浮液。

从小鼠颈动脉制备用于单细胞测序的单细胞悬液

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

溶解度低和细胞分布较大是血管组织解离的主要困难。我们的两步或消化方法非常适合颈动脉组织解离，可用于为其他血管准备单细胞怀疑，只需稍作修改，对心血管研究非常有帮助。该方案适用于在颈动脉中获得多种细胞类型，无需特殊设备即可轻松重复。未来，我们的目标是利用单细胞离子测序发现的细胞亚类进行心血管疾病的靶向治疗。

preparation-single-cell-suspension-from-mouse-carotid-arteries-for

Research

JoVE Journal

Biology

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

2.6K 次观看.  Soochow University. 溶解度低和细胞分布较大是血管组织解离的主要困难。我们的两步或消化方法非常适合颈动脉组织解离，可用于为其他血管准备单细胞怀疑，只需稍作修改，对心血管研究非常有帮助。该方案适用于在颈动脉中获得多种细胞类型，无需特殊设备即可轻松重复。未来，我们的目标是利用单细胞离子测序发现的细胞亚类进行心血管疾病的靶向治疗。

视频: 作者聚焦：血管组织解离和探索用于靶向治疗的单细胞亚簇 

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

科研

生物学

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

从小鼠神经组织产生单细胞悬浮液

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

从小鼠颈动脉管腔中分离内皮细胞以进行单细胞多组学实验

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

用化学发光法实时检测水稻免疫反应中活性氧的产生

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

尼罗罗非鱼肠单细胞悬液制备用于单细胞测序

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

植物细胞类型的分离和转录组分析

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...