Name: quality-controlled sputum analysis by flow cytometry
Uploaded: 2021-08-09T13:00:00
Description: Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

我们要感谢大卫·罗德里格斯（David Rodriguez）对人物准备工作的协助。痰液样本在UT Health San Antonio流式细胞术共享资源设施的BD LSR II上运行，由UT Health，NIH-NCI P30 CA054174-20（UT Health的CTRC）和UL1 TR001120（CTSA授权）提供支持。

痰液处理的所有步骤都在带有适当个人防护设备的生物安全柜中进行。
1. 开始痰液解离前的试剂制备
<ol><li>在冰上解冻1%多聚甲醛（PFA），每个样品25 mL，并保持冷藏直至使用。 注意：PFA通过吸入和皮肤接触而有毒。根据制造商的说明准备固定剂，并在-20°C下冷冻在25 mL等分试样中直至使用。</li>
	<li>近似样品的重量并解冻足够的0.1%二硫磷脂醇（DTT）用于步骤2.2，并将其?...

痰液的细胞含量包括种类繁多的宽范围细胞，往往伴有大量碎片37。此外，痰液分析需要质量控制，以确认样本是从肺部而不是口腔收集的38。因此，通过流式细胞术分析痰液并不像血液那样简单，例如，血液释放出更清洁和均匀的细胞悬浮液。该协议已经解决了所有这些问题：提供使用特定尺寸的微球的仪器设置，以确保可以检测到最小和最大的细胞群，门控...

流式细胞仪硬件和软件的技术进步使得同时识别许多不同的细胞群成为可能1，2，3，4。例如，流式细胞仪在造血细胞研究中的应用使人们对免疫系统2和造血系统的细胞层次结构5有了更好的了解，并区分了多种不同血癌的诊断方法6，7，8。虽然大多数痰细胞起源于造血9，10，11，但流式细胞术尚未广泛应用于用于诊断目的的痰液分析。然而，一些研究表明，评估痰液（最重要的细胞亚群）中的免疫细胞群可能对诊断和/或监测哮喘和慢性阻塞性肺病（COPD）等疾病有很大帮助12，13，14，15。此外，可用于流式细胞术的上皮特异性标志物的存在允许询问痰液中以下最重要的细胞亚群，肺上皮细胞。
除了能够分析不同组织起源的许多不同细胞群之外，流式细胞仪还可以在相对较短的时间内评估大量细胞。相比之下，基于载玻片的细胞学分析类型通常需要高度专业的人员和/或设备。这些分析可能是劳动密集型的，这导致只有一部分痰液样本被分析16。
三个关键问题限制了痰液在流式细胞术中的广泛使用。第一个问题涉及痰液的收集。痰液通过喘息咳嗽收集，将粘液从肺部排出到口腔中，然后吐入收集杯中。由于粘液穿过口腔，因此SEC污染的可能性很高。这种污染使标本分析复杂化，但如本研究所示，该问题在流式细胞仪平台上很容易纠正。
不是每个人都能自发产生痰液;因此，已经开发了几种设备来帮助以非侵入性方式收集痰液17。雾化器就是这样一种装置，已被证明可以产生可靠的痰液样本18，19，20。虽然雾化器是一种非常有效的非侵入性收集痰液的方法，但其使用仍然需要在有专业人员的医疗机构进行设置21。相比之下，手持设备，如肺笛22，23，24 和无伴奏合唱16，25 ，可以在家中使用，因为它们非常人性化。这些辅助设备既安全又经济高效。
对于我们来说，阿卡贝拉的效果一直比肺笛16更好，因此，阿卡贝拉装置已被选择用于痰液收集。决定使用3天的收集样本，因为使用痰液的主要目的是开发肺癌检测测试16。已经表明，与1天或2天的样本相比，3天的样本增加了肺癌检测的可能性26，27，28。然而，对于不同的目的，其他的痰液收集方法可能更可取。如果使用与此处描述的方法不同的痰液收集方法，建议仔细滴定用于流式细胞术分析的每种抗体或染料;关于不同的痰液收集方法如何影响流式细胞术的靶向蛋白质的数据很少。
抑制使用痰液进行诊断的热情的第二个问题是细胞数量，主要与流式细胞术有关。问题是收集足够的活细胞进行可靠的分析。两项研究表明，在辅助装置的帮助下，通过非侵入性方法收集的痰液样本含有足够的活细胞，可用于临床诊断或研究研究16，24。然而，这两项研究都没有解决流式细胞术的细胞数量问题。
对于构成该方案基础的研究，根据每个研究地点批准的机构指南，从患肺癌高风险的参与者中收集痰液样本。高危参与者被定义为55-75岁之间，吸烟30包年，过去15年内没有戒烟。向患者展示了如何根据制造商的说明使用阿卡贝拉装置29 ，并在家中连续三天收集痰液。样品保存在冰箱中，直到最后一次收集。在最后一个收集日，样品与冷冻冷藏袋一起连夜运往实验室。样品在收到当天被加工成单细胞悬浮液。使用这种痰液收集方法，可以获得足够的活细胞，以进行可靠的流式细胞术分析。
最后，与之前的细胞数问题相关的是如何将痰细胞从其粘液环境中释放出来的问题。如何保持细胞存活并产生不会堵塞流式细胞仪的单细胞悬浮液？基于Pizzichini等人30 和Miller等人的初步工作，该协议描述了一种简单可靠的方法，用于将痰液加工成适合流式细胞术分析的单细胞悬浮液。该方法在流式细胞术中使用了完善的指南32，33，34 来开发有效的抗体标记策略，以识别痰液中的造血细胞和上皮细胞，并提供仪器设置，质量控制措施和分析指南，在流式细胞术平台上标准化痰液分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% Paraformaldehyde Flow-Fix</td>
			<td>Polysciences</td>
			<td>25037</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;M nylon cell strainers, Falcon #352360</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M NaOH</td>
			<td>EMD</td>
			<td>SX0593-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical falcon tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 anti-human CD19</td>
			<td>BioLegend</td>
			<td>302219</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 anti-human CD3</td>
			<td>BioLegend</td>
			<td>300415</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 anti-human cytokeratin</td>
			<td>BioLegend</td>
			<td>628608</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 PanCK, CD3, and CD19 Isotype</td>
			<td>BioLegend</td>
			<td>400129</td>
			<td></td>
		</tr>
		<tr>
			<td>BV510 anti-human CD45</td>
			<td>BioLegend</td>
			<td>304036</td>
			<td></td>
		</tr>
		<tr>
			<td>CD66b FITC isotype</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td></td>
		</tr>
		<tr>
			<td>CompBead Plus Compensation Beads</td>
			<td>BD Biosciences</td>
			<td>560497</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Polystyrene dispoable sterile bottle 250 mL</td>
			<td>Fisher Scientific</td>
			<td>09-761-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Polystyrene dispoable sterile bottle 500 mL</td>
			<td>Fisher Scientific</td>
			<td>09-761-10</td>
			<td></td>
		</tr>
		<tr>
			<td>CS&#38;T beads</td>
			<td>BD Biosciences</td>
			<td>655051</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD66b</td>
			<td>GeneTex</td>
			<td>GTX75907</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Stain</td>
			<td>BD Biosciences</td>
			<td>564406</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowCheck</td>
			<td>Beckman Coulter</td>
			<td>A69183</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowSet</td>
			<td>Beckman Coulter</td>
			<td>A69184</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Fisher Scientific</td>
			<td>14-175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>NAC</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>NIST Beads, 05 &#956;M</td>
			<td>Polysciences</td>
			<td>64080</td>
			<td></td>
		</tr>
		<tr>
			<td>NIST Beads, 20 &#956;M</td>
			<td>Polysciences</td>
			<td>64160</td>
			<td></td>
		</tr>
		<tr>
			<td>NIST Beads, 30 &#956;M</td>
			<td>Polysciences</td>
			<td>64170</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD45</td>
			<td>BioLegend</td>
			<td>304039</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 anti-human EpCAM</td>
			<td>BD Biosciences</td>
			<td>565399</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 CD206/EpCAM Isotype</td>
			<td>BD Biosciences</td>
			<td>562292</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CR594 anti-human CD206</td>
			<td>BD Biosciences</td>
			<td>564063</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>EMD</td>
			<td>SX0445-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution, 0.4%</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
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quality-controlled sputum analysis by flow cytometry

痰液广泛用于研究细胞含量和其他微环境特征，以了解肺部的健康，传统上使用基于细胞学的方法进行分析。它的效用有限，因为阅读幻灯片非常耗时，需要高度专业化的人员。此外，广泛的碎片和过多的鳞状上皮细胞（SEC）或脸颊细胞的存在往往使样本不足以诊断。相比之式细胞术允许对细胞群进行高通量表型分析，同时排除碎片和 SEC 。
这里介绍的方案描述了一种将痰液解离成单细胞悬浮液，抗体染色和固定细胞群以及在流式细胞术平台上获取样品的有效方法。这里介绍了一种门控策略，描述了排除碎片，死细胞（包括SEC）和细胞二倍体。此外，这项工作还解释了如何基于分化簇（CD）45阳性和阴性群体分析活的单个痰细胞，以表征造血和上皮谱系亚群。通过识别肺部特异性巨噬细胞作为样本来自肺部而不是唾液的证据，还可以提供质量控制措施。最后，已经证明该方法可以通过提供在三个流式细胞仪上分析的同一患者的痰液图谱应用于不同的细胞术平台;Navios EX，LSR II和Lyric。此外，可以修改该协议以包括感兴趣的其他细胞标记物。这里提出了一种在流式细胞术平台上分析整个痰液样本的方法，该方法使痰液适合于开发肺病的高通量诊断。

该协议是在考虑临床实验室环境的情况下制定的。在协议开发过程中，重点是简单性，效率和可重复性。研究发现，处理痰液中最耗时的步骤是计数细胞。因此，该协议的设置方式使得痰液处理和细胞标记可以独立于细胞计数进行而不会浪费时间。然后，可以在抗体标记孵育期间获得准确的细胞计数，这对于适当稀释样品以进行无障碍运行仍然是必需的。
该协议使用痰液重?...

Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

Quality-Controlled Sputum Analysis by Flow Cytometry

该协议描述了一种将痰液解离为单细胞悬浮液的有效方法，以及随后在标准流式细胞术平台上对细胞亚群的表征。

通过流式细胞术进行质量控制痰液分析

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

该协议解决了痰液处理过程中遇到的常见挑战，这些挑战在过去使得流式细胞术难以用于高通量临床诊断目的。该协议旨在通过使用痰量来确定抗体和染料染色量来节省时间。在染色孵育期间，可以对下游步骤进行细胞计数。重要的是要实现准确的细胞计数，以确定正确的再悬浮液体积，以防止在流式细胞仪上运行时出现任何问题。该协议可以提供对研究肺部健康的研究领域的见解。例如，可以研究COPD，哮喘，肺癌或COVID的影响等疾病。演示该程序的将是Michael Lai博士，他是我实验室的研发科学家。首先，称量痰液样本以确定解离试剂的体积。根据痰液重量将样品转移到适当大小的血管中，然后每克0.5%NAC加入一毫升，每克0.1%DTT加入四毫升。在室温下以最大速度涡旋样品15秒，以最大速度摇摆15分钟。用4X Hanks平衡盐溶液或HBSS稀释该样品，以中和NAC和DTT。快速涡旋后，在室温下摇动样品五分钟。通过100微米尼龙网状细胞过滤器过滤细胞悬浮液到一个或多个50毫升锥形离心管中，以产生单细胞悬浮液。将细胞向下沉淀。吸出上清液后，将沉淀物混合在一个15毫升的锥形管中，并在相同的条件下用HBSS洗涤。将细胞沉淀重悬于由痰液样品的初始重量确定的缓冲液体积中。从细胞悬浮液中取出等分试样，将10微升痰液稀释液与30微升0.4%台盼蓝混合，然后装入血细胞计数器的计数室进行活/死细胞计数。在补偿管中标记痰液。如文中所示，将HBSS抗体和染料添加到每个痰细胞管和补偿管中。如文中所述，将痰细胞添加到测定管中，并将补偿珠添加到补偿管中。将所有管子在冰上避光35分钟。然后，在用冰冷的HBSS填充管后，以800倍G在4摄氏度下离心10分钟，对于补偿管，吸出上清液尽可能靠近颗粒并轻拂颗粒以松动。接下来，将0.5毫升冷HBSS加入补偿管中，并将管子储存在冰上，在轻度保护区，直到需要流式细胞术分析。然后从未染色的痰液、同种型、血液和上皮管中吸出上清液。通过轻拂管子松开颗粒。向未染色的和同种型管中加入2毫升1%PFA，向血液和上皮管中加入10毫升。将管子以轻度保护的方式在冰上孵育30分钟，然后以最大速度快速涡旋，然后再次孵育30分钟。然后用冰冷的HBSS填充管子。接下来，在4摄氏度下离心管10分钟，以1，600倍G.A吸出尽可能多的上清液而不干扰细胞沉淀，并用手指轻拂管以松开细胞，然后将200微升冷的HBSS加入未染色的和同种型管中。根据总细胞计数计算并添加血液和上皮管重悬所需的HBSS体积。将所有样品储存在补偿管中，以防四摄氏度的冰上避光，直到进行流式细胞术分析。对正在使用的流式细胞仪应用适当的启动程序。使用美国国家标准与技术研究院或 NIST 磁珠的混合物，确保将正向散射和侧向散射电压设置为将 NIST 磁珠放置在跨图中，而不会将磁珠放置在离轴太近的位置。对于 LSRII，将流速设置为中等流量，对于 Navios EX，将流速设置为高。调整用于散射和荧光参数的每个参数的电压，以按比例放置细胞群。使用带有门控策略的数字作为指导，相应地调整电压。首先获取未染色的痰液样本的数据，然后获取同种型染色样本的数据，然后获取血管和上皮管。痰液重量用于测定用于染色的抗体或染料体积。痰量与细胞产量的相关性不强。痰液样本分为4个重量类，在细胞产量分布中聚类。选择在平台相上以具有最高染色指数的浓度滴定使用的每种抗体作为工作浓度。在所有重量类别中，平均SEC污染小于20%，显示活细胞的百分比。介绍的是用于分析的门控策略。NIST珠子用于设置一个门，该门应用于痰液样本以消除碎片。宽度门进一步消除了小碎片。活性染料用于消除死细胞，而单线态门去除双偶氮。抗CD45抗体标记允许将活的单个痰细胞分离到血细胞和非血细胞室中。比较了从Navios EX，LSRII和Lyric获得的配置文件。图中显示了用于消除碎片、死细胞、双联物以及比较血液和非血液剖面的门控策略。这项技术使我们能够应用这些方法来检测肺癌风险，以用作非侵入性临床试验。

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SCIENTISTS IN ACTION

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

科研

医学

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

分子探针的优化，以确定细胞死亡的光合生物（<em&gt;铜绿微囊藻</em&gt;）使用流式细胞仪

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

痰液诱导和实验室处理方法

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.