Name: fluorescence-activated cell sorting for the isolation of scleractinian cell populations
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NTK希望表彰迈阿密大学自然科学和工程研究奖，以表彰他为这项研究提供资金。BR感谢亚历克斯和安·劳特巴赫为比较和进化免疫学实验室提供资金。BR的工作得到了以色列科学基金会（ISF）号码：1416/19和2841/19以及HFSP研究赠款RGY0085/2019的支持。我们要感谢詹娜·科热克巴耶娃和迈克·康纳利的技术援助。我们还要感谢迈阿密大学、米勒医学院在西尔维斯特综合癌症中心的流动细胞测量共享资源，感谢他们使用FACS细胞计和香农·赛格获得技术支持。

1. 通过喷枪和压缩机从珊瑚骨架分离组织
注：在冰上执行步骤，用手套保护双手。
<ol><li>通过连接空气压缩机、软管和喷枪来组装喷枪套件（图2）。将压力表设置为 276×483 kPa 之间。 注： 本研究所用的推荐压缩机和空气软管预设为最大压力为 393 kPa。在此 276-483 kPa 范围之外使用可能会导致细胞从骨架中去除不足或细胞膜破裂。此范围可能因每个物?...

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该协议改编自罗森塔尔等人18号，用于识别和隔离P.Damicornis细胞。该方法侧重于通过检查细胞内在因素（包括相对细胞大小、相对细胞粒度、细胞自荧）和完整细胞膜的存在，过滤样品以去除碎屑、非活细胞和共生细胞承载细胞的过程。这些技术可以应用于其他珊瑚物种。然而，FACS浇注策略可能因细胞种群特征的物种特异性差异而异。
FACS对了解珊瑚?...

珊瑚礁是地球上最重要的生态系统之一。它们通过提供鱼类和无脊椎动物的关键栖息地促进生物多样性，并通过旅游业1提供粮食和经济生计，对维持人类社区至关重要。作为珊瑚礁的主要建设者，珊瑚动物（Phylum：Cnidaria）也通过创建大型碳酸钙框架来缓解海浪和风暴破坏2，帮助沿海社区。
珊瑚作为成人是分离动物，容纳广泛的内生生物伙伴，包括病毒，古生物，细菌，蛋白物，真菌，最值得注意的是，藻类二丁那胶酸盐家族共生体3的成员。环境的变化可能导致这个社区的不平衡，往往导致疾病爆发和珊瑚白化，其中共生共生共生体被逐出珊瑚群落，从而消除珊瑚的主要营养来源。这两种情况经常导致珊瑚宿主44、5、65,6的死亡。人为诱发的压力因素的影响，如快速的气候变化，正在加速大规模珊瑚死亡事件，导致全球珊瑚礁减少7。
最近，已经开发了许多不同的方法来帮助减轻珊瑚礁的损失。这些方法包括在现有珊瑚礁上种植珊瑚，使用耐热基因型进行基因交叉，以及对珊瑚88、99内托管的微生物和共生群落进行细胞操作。尽管作出了这些努力，关于珊瑚细胞多样性和细胞功能10、11、12、13,12,13仍不为人所知。10,要了解珊瑚生物在规范和紧张条件下行为的方式，必须全面了解珊瑚细胞类型的多样性和细胞功能。对细胞多样性和基因功能如何耦合的理解将得益于最大限度地提高恢复和保存效率的努力。
以前关于细胞多样性和功能的研究主要集中在组织学研究和全组织RNA取样14，15，16，17。14,15,16,17为了更详细地了解珊瑚中的特定细胞类型功能，需要有分离活珊瑚细胞特定种群的方法。这已经成功地在非经典模型生物体中，通过荧光活性细胞分选（FACS）流动细胞测量技术18。FACS 利用调谐到不同波长的激光组合来测量单个细胞级别的不同内源细胞特性，如相对细胞大小、细胞粒度和自荧光。此外，细胞可能用荧光标记的化合物标记，以测量特定的，所需的属性18，19。18,
到目前为止，流细胞学在珊瑚细胞中的应用主要是利用其强、天然自氟化20、21、22,22的共生共生共生生物和其他细菌种群进行分析。20,FACS也被用来估计珊瑚基因组的大小，通过使用荧光DNA标记信号比较参考模型有机体细胞23，24。23,FACS的有效应用提供了三种不同的工具，对细胞生物学研究有用：1） 单细胞的形态和功能描述;2） 识别、分离和分离下游研究的特定细胞群;和3）单细胞水平功能测定分析。
各种外源荧光标记物的开发和应用，用于研究珊瑚细胞，目前仍有待开发。此类标记可能包括标记的蛋白质、酶标记的基质或对其他化合物的荧光反应。这些标记可用于识别具有唯一属性的细胞类型，例如突出显示产生不同量特定细胞室特征（如隔离体）的单元格。另一个例子是使用荧光标记的珠子来功能识别能够进行噬菌体细胞的细胞，或吞没靶向病原体25。在免疫反应中活跃的细胞群，在吞没这些外源应用的珠子后，可以很容易地识别出。虽然传统的组织学方法需要保存的组织和几个小时来近似珠类吞没的阳性细胞的百分比，但基于FACS的病原体吞没功能测定可以在分离的活细胞上相对快速地进行。除了研究细胞对压力的特异性反应外，该技术还有可能阐明基因特异性表达，并阐明细胞类型的进化和发育历史，这些细胞类型完全为细胞类型所独有，这些细胞类型完全为细胞类型所独有，如卡利波和神经细胞。
最近，我们对30多个细胞标记进行了密集筛选，从而确定了24个能够标记珊瑚细胞的标记，其中16个可用于区分独特的种群18，使它们成为分化集群（CD）。在这里，我们描述了在波西洛波拉达米科尼斯的珊瑚细胞分离过程，从从碳酸钙骨架中取出细胞到用FACS识别和隔离特定细胞群（图1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Airbrush Kit &#38; Compressor</td>
			<td>TCP Global</td>
			<td>ABD KIT-H-SET</td>
			<td>Paasche H Series Single-Action Siphon Feed Airbrush Kit with Master TC-20 Compressor &#38; Air Hose</td>
		</tr>
		<tr>
			<td>BD FACSAria II</td>
			<td>BD</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Cutters</td>
			<td>Bulk Reef Supply</td>
			<td>205357</td>
			<td>Oceans Wonders Coral Stony Bone Cutter</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td>40 um; BD Falcon; individually wrapped; sterile; nylon</td>
		</tr>
		<tr>
			<td>CellRox Green</td>
			<td>Life Technologies</td>
			<td>C10444</td>
			<td>2.5 mM in DMSO; Excitation/Emission: 485/520 nm</td>
		</tr>
		<tr>
			<td>Collection bag</td>
			<td>Grainger</td>
			<td>38UV35</td>
			<td>Reloc Zippit 6&#34;L x 4&#34;W Standard Reclosable Poly Bag with Zip Seal Closure, Clear; 2 mil Thickness</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>10mg in H2O; Excitation/Emission: 358/461 nm</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-100ML</td>
			<td>Heat-inactivated at 57 &#176;C for 30 minutes</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Deep Red</td>
			<td>Life Technologies</td>
			<td>L12492</td>
			<td>1mM in DMSO; Absorption/Emission: 647/668 nm</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Phophate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td>pH 7.4; 10X</td>
		</tr>
		<tr>
			<td>Round-bottom tubes</td>
			<td>VWR</td>
			<td>352063</td>
			<td>5 mL Polypropylene Round-Bottom Tube</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td>1 mL BD Luer-Lok Syringe sterile, singe use polycarbonate</td>
		</tr>
	</tbody>
</table>

fluorescence-activated cell sorting for the isolation of scleractinian cell populations

由于人为压力因素，珊瑚礁受到威胁。珊瑚对这些压力器的生物反应可能发生在细胞水平上，但这些机制并不十分了解。为了研究珊瑚对压力器的反应，我们需要分析细胞反应的工具。特别是，我们需要有助于应用功能检测的工具，以更好地了解细胞群对压力的反应。在目前的研究中，我们使用荧光激活细胞分拣（FACS）来分离和分离石珊瑚中不同的细胞群。该协议包括：（1） 珊瑚组织与骨骼分离，（2） 创建单个细胞悬浮液，（3） 使用各种标记标记标记珊瑚细胞进行流细胞测定，以及 （4） 门控和细胞分拣策略。这种方法将使研究人员能够在细胞水平上研究珊瑚，以便对不同的细胞群进行分析、功能测定和基因表达研究。

总体而言，该协议非常有用，因为它有助于识别和收集可用于功能分析的活珊瑚细胞种群。工作流程开始于珊瑚组织与底层碳酸钙骨架的机械分离（图1）。这是最重要的初始步骤之一，因为不当技术会导致高细胞死亡率，并会产生大量碎片。不建议进行酶分离，因为它可能导致组织剪切增加和细胞死亡率高。使用喷枪进行机械分离可减少这些挑战（...

Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations at JoVE.com

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

珊瑚创造了对人类和海洋生物都重要的生物多样性生态系统。然而，我们仍然不明白许多珊瑚细胞的全部潜力和功能。在这里，我们提出了为石珊瑚细胞群的分离、标记和分离而开发的协议。

荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

该协议有助于在以前不可能确定的描述水平上识别和隔离活珊瑚细胞种群。这种技术的主要优点是，它允许访问一个细胞特异性水平，到目前为止，已经基本上无法实现。展示这个程序的将是山农·赛格博士，一个研究助理，来自西尔维斯特综合癌症中心的流细胞学共享资源。要设置活珊瑚细胞分拣的流动细胞仪，请在流式细胞仪软件中打开一个新的项目模板，并在激光面板中选择合适的激光器进行分析。然后，在适当的图中创建侧散点图与前进散点图，以设置浇注和分析策略。对于活珊瑚细胞的流动细胞测量分析和分拣，将控制未污染的细胞样本加载到细胞仪的样品室中，然后开始读取过程。由于珊瑚细胞特别脆弱，请保持细胞计的低气压，以防止细胞解。在散点图中，调整光倍增器电压以居中点并开始记录事件。在前进与侧散射图中，在向前散射 x 轴上 10 到 4 个标记周围的单元格周围创建一个栅极。用染色细胞的第一个样本替换控制样本，并在暂停前记录大约 10，000 个细胞。然后对染色样本组唯一的事件进行门控。要对活珊瑚细胞进行排序，在选择流式细胞仪软件中感兴趣的细胞群后，将一个包含250至500微升适当收集溶液的微离心管放入细胞计收集室，供每个细胞量采集室进行排序。一旦经过适当的时间来清除碎片，开始整理所需的种群，收集从1万到数百万个细胞的任何地方。每次使用新的污渍、物种或分拣器时，将至少2万个感兴趣的细胞分类成500微升染色介质，以便对感兴趣的群体进行纯度检查，并重新分析分拣细胞，以确认事件正在用于初始分拣的大门内读取。对于体外研究，将分类的细胞储存在冰上，直到它们被转移到培养箱或灭菌环境。通过在向前和侧面散射图上创建栅极选择，引入与珊瑚细胞不具有相同形状或粒度的其他非细胞粒子。通过使用直方图比较未染色和染色细胞悬浮液的 DAPI 信号，并找出高 DAPI 表达细胞，可以排除死细胞。同时，通过对远红色通道中信号高的细胞进行浇注，可以去除自荧光共生细胞。经过这个迭代的浇注过程，剩余的细胞代表缺乏共生亚的完整活珊瑚细胞种群。然后，这些细胞可以通过染色（如活性氧物种活性和/或糖体含量）的染色，分类为不同的细胞子组。请记住，某些细胞比另一些细胞更脆弱，并尽可能小心地运行此过程。一旦分离出特定的细胞，它们就可用于x-vivo功能测定，如建立细胞培养和创建转录体配置文件。

Research

JoVE Journal

Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

分离和造血干细胞移植（造血干细胞）

科研

生物学

Fluorescence Activated Cell Sorting of Plant Protoplasts

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

排序<em&gt;链霉菌</em&gt;细胞沉淀用一个复杂的对象参数分析仪和分选机

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...