这项工作得到了以色列科学基金会（Grant 119/16）和IMoh资助（3-15656）对IKG的支持。R.S. 由 Kreitman 奖学金支持。

.ast 文件以 鼠李糖乳杆菌 GG （LGG） 的模板为例，在 补充编码文件 1 中提供。
1. 培养基制备
<ol><li>按照制造商的说明（55 g 在 1 L 去离子水中）和 1.5% （w/v） 琼脂制备乳酸杆菌 MRS 琼脂平板（参见 材料表）。高压灭菌后，培养基可直接使用或可在4°C下储存。</li>
	<li>准备 50%（15 g 在 1 L 去离子水中）胰蛋白酶大豆肉汤 （TSB）（参见 材料表），TSB 补充有 1% （w/v） D-（+）-葡萄糖和 1% （w/v） D-（+）-棉子糖，然后高压灭菌以灭菌培养基。最好将其储存在4°C以避免污染。</li>
	<li>对于含有葡萄糖的缓冲培养基，制备补充有 1% （w/v） D-（+）-葡萄糖、5 mM 磷酸二氢钾和氢二钾和 100 mM MOPS（3-（N-吗啉基）丙烷磺酸，参见 材料表）pH 7 的 TSB。然后高压灭菌以对培养基进行灭菌。</li>
</ol>2. 样品的制备
注意：此步骤涉及评估细胞聚集对饮食中可发酵或不可发酵糖的反应。样品制备过程的示意图如图 1 所示。
<ol><li>在乳酸杆菌MRS肉汤板（1.5%琼脂）上从冷冻甘油原液（50%甘油）中划线乳酸杆菌科菌株。 注：在此介绍的示例实验中，使用了 嗜酸乳杆 菌 （ATCC 4356）、 干酪乳杆 菌 （ATCC 393）、干酪乳酪杆菌 副干酪亚种 （ATCC BAA-52）、 植物乳 杆菌 （ATCC 8014） 和 鼠李糖乳杆 菌 GG （ATCC 53103） （LGG） 益生菌菌株。为了将该方法扩展到厚壁菌门的其他益生菌成员，选择了 凝结芽孢杆 菌（ATCC 10545）。</li>
	<li>在静态条件下在37°C下生长细胞过夜。</li>
	<li>将5mL乳酸杆菌MRS肉汤与单个菌落接种，并在37°C的静态条件下生长过夜。</li>
	<li>在 3 mL 50% 胰蛋白酶大豆肉汤 （TSB） 中稀释上一步 （1：100） 的细胞培养物，TSB 补充有 1% （w/v） D-（+）-葡萄糖，或 TSB 补充有 1% （w/v） D-（+）-棉子糖。</li>
	<li>在静态条件下在37°C孵育过夜。</li>
	<li>通过在培养基中均匀混合细胞来制备样品，并从步骤 2.5 开始轻轻涡旋细胞培养管。轻轻地倒置试管，直到完全混合。将 200-300 μL 转移到 1.5 mL 微量离心管中。 注意：必须不要对样品进行超声处理，以免破坏聚集体。在从步骤 3.4 到 3.10 的数据采集过程中，可能会出现样本过于集中的情况。如果观察到样品运行非常缓慢或根本不运行，则可以使用适当的新鲜培养基稀释样品。</li>
</ol>3. 数据采集
<ol><li>根据制造商提供的说明设置成像流式细胞仪（参见 材料表）。</li>
	<li>将 785 nm 激光器设置为 5 mW 进行暗场测量（相当于传统流式细胞术中的侧向散射，缩写为 SSC）。使用数值孔径 （N.A） 为 0.9 的 60 倍镜头，选择 高灵敏度，并将速度设置为 低。</li>
	<li>在收集任何数据之前，请确保校准磁珠在 SSC 通道中的流动稳定性。单击“Fluidics”框中的 播放 按钮，然后检查磁珠图像的质量。珠子图像应看起来清晰明了。</li>
	<li>按下 加载 按钮，将装有样品的试管（来自步骤 2.6）放入支架中。</li>
	<li>在“工作区”框中创建一个新的散点图。单击 “新建散点图 ”，然后在明场通道中选择与 SSC 通道强度相对应的区域。排除与样品一起在仪器中运行的校准珠。 注意：可以通过仅加载带有缓冲液的样品并识别区域与上的磁珠 来排除磁珠。SSC图。然后使用 “创建多边形区域 ”按钮在珠子群周围绘制一个门。</li>
	<li>在“采集设置”框中，输入 样本名称，指定数据存储位置，选择要记录的群体（没有磁珠的所有细胞），并设置要收集的事件数。通常，10,000-20,000 个事件就足够了。</li>
	<li>单击位于“采集”框中的 “录制 ”按钮，一旦达到指定的事件数，采集过程将自动结束。</li>
	<li>单击 “返回 ”按钮卸下试管，并在小窗口中出现提示时从支架中取出试管。</li>
	<li>对每个样品重复步骤 3.5-3.9。</li>
	<li>保存模板以保持数据采集的均匀性，以便后续重复。</li>
</ol>4. 数据分析
<ol><li>测量来自总体的自动聚合的百分比 （%）。<ol><li>使用IDEAS软件分析在成像流式细胞仪上采集的数据（参见 材料表）。</li>
		<li>通过将 （.rif） 文件加载到分析软件中，从原始文件 （.rif） 创建数据分析文件 （.daf）。单击“ 文件 ”按钮，然后打开所需的 （.rif） 文件。在打开的窗口中，单击“ 使用采集分析 ”，然后单击“ 确定”。</li>
		<li>在明场通道中创建梯度 RMS（图像对比度和焦点的测量）直方图，以识别聚焦事件。在分析区域中，单击 “新建直方图 ”按钮。在“新建直方图”窗口中，从采集中选择要分析的总体，然后在“x 轴特征”中，选择明场通道的 梯度 RMS 。<ol><li>通过单击分析区域中的 “创建线区域 ”按钮来放置门，以排除非聚焦事件（图2A）。 注意：通常，“聚焦”线区域应包括 80%-90% 具有最高梯度 RMS 分数的事件，但应为每个实验设置确定“聚焦”单元格的特定阈值。</li>
		</ol></li>
		<li>创建面积 （μm2） 与明场通道的纵横比（宽度除以最佳拟合椭圆的长度）的散点图。在分析区域中按 新建散点图 按钮。在“新建散点图”窗口中，从步骤 4.1.3 中选择 聚焦 人口。<ol><li>为 x 轴特征选择明场通道的 面积 ，为 y 轴特征选择明场通道的 纵横比 。该散点图允许对聚焦群体进行门控，以区分单峰和小聚集体事件与较大的微生物聚集体和链。</li>
		</ol></li>
		<li>在此散点图上，根据聚合事件群体的面积值，使用 “创建矩形区域 ”按钮（或 “创建多边形区域”）绘制一个门，并为单峰和小聚合群体绘制另一个门（图 2B）。 注意：在某些情况下，从单峰中分离小聚集体可能具有挑战性，因此它们可以合并到同一个浇口中。这同样适用于较大的聚集体和链（图 3）。</li>
		<li>手动查看和评估每个闸门内的事件图像，以验证闸门策略的可靠性。如果需要，请调整闸门的面积值。通过从“人口”下拉菜单中选择已审查的人口来执行此操作。 注意：设门没有特定的面积值，因为它可能会根据被分析细菌的特性而变化。</li>
		<li>将数据分析另存为模板。单击 “文件 ”选项卡，选择“ 另存为 Template.ast”，然后打开同一模板下的下一个示例 （.rif） 文件，以创建数据分析文件 （.daf）（支持 .ast 文件作为 LGG 的示例）。</li>
		<li>创建一个统计信息表以枚举关键事件以供进一步分析。单击“ 报告 ”选项卡，然后单击“ 定义统计信息报告”。</li>
		<li>在新窗口中，单击“ 添加列”。添加单峰/聚合计数和 %门控统计信息。在“统计信息”下，选择“ %门控/计数”，然后在所选总体下，选择 单峰/聚合。单击“ 添加统计信息 ”以将统计信息添加到列表中并将其另存为模板。</li>
		<li>单击“ 生成统计报告 ”，然后选择统计模板和 （.daf） 文件进行分析。</li>
	</ol></li>
	<li>测量聚集体的尺寸分布。<ol><li>要分析聚集事件的平均大小，请绘制步骤4.1.5中聚集事件群体的面积（μm2）直方图（图2C）。</li>
		<li>将数据分析另存为模板。单击“ 文件 ”选项卡，选择“ 另存为模板”，然后在数据分析文件 （.daf） 的同一模板下打开下一个示例 （.rif） 文件。</li>
		<li>重复步骤 4.1.7-4.19。对于聚合的大小，请在统计数据下选择 均值 ，然后在所选总体下选择 聚合 。在“要素”下，选择“明场通道 的区域 ”。单击“ 添加统计信息 ”以将统计信息添加到列表中并将其另存为模板。</li>
		<li>重复步骤 4.1.9 以生成统计报告。</li>
	</ol></li>
</ol>

使用成像流式细胞术定量分析微生物自聚集

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流式细胞术是一种广泛使用的方法，用于量化真核细胞中的荧光强度，但由于细菌细胞的尺寸较大或聚集体较小，它可能无法为细菌细胞提供准确的测量结果。这些因素会显著影响自聚集的精确定量和不同条件下聚集体形成的基础水平。为了解决这个问题，采用成像流式细胞术 （IFC） 来更好地解析碳水化合物如何影响益生菌聚集5。IFC 通过收集每个样本的大量数字图像并提取基...

细菌自聚集被认为是生物膜形成的主要步骤。在此过程中（有时也称为自凝集或絮凝），相同类型的细菌形成多细胞团块，最终沉淀在培养管底部或附着在其靶组织或表面1。
自聚集是一种广泛观察到的现象，迄今为止已在革兰氏阴性病原体中得到证实，例如机会性病原体鲍曼不动杆菌 2、牙科病原体放线菌聚集杆菌 3 和新出现的病原体 Burkholderia pseudomallei4。在几种益生菌革兰氏阳性菌株中也描述了自聚集 5,6,7,8。在嗜酸乳杆菌 （L.） 中，自聚集部分由 S 层蛋白介导，并与与木聚糖7 的粘附相关。同样，我们发现葡萄糖依赖性自聚集与益生菌物种 Lacticaseibacillus rhamnosus GG、Lacticaseibacillus casei、L. acidophilus、Lacticaseibacillus paracasei 和 Lactiplantibacillus plantarum5 的粘附特性（通过粘蛋白粘附来判断）之间存在相关性.自聚集的增加很可能反映了细胞粘附素表达对葡萄糖及其分解代谢物的反应变化9。虽然自聚集的分子机制仍有待确定，但已经表明，该过程改变了聚集细菌的表型，并赋予它们对环境压力源1 的增强耐受性，并增加了对群体感应分子的敏感性10。
已经使用了几种方法来测量自聚合;一种实验方法是让培养物在狭窄的培养管中静态放置给定时间。对照培养物保持浑浊，而自聚集培养物将沉淀在试管底部。一种更定量的方法通过沉降或沉降测定法测量自聚集11。
近年来，流式细胞术也越来越多地用于研究细菌自聚集。该方法适用于分析大小约为 0.5 至 1000 μm 的颗粒。将单个细菌或形成的聚集体悬浮在液体中，送入液流中，并且可以逐个检测11。记录前向散射光可以测量细胞或聚集体的相对大小。它相对快速和直接，但无法检测多个参数，例如聚集体大小或聚集体中的平均单元格数。因此，这种方法可以在显微镜上进行补充，允许检查更多的参数12。然而，传统的显微镜检查非常耗时，因此限制了测试样品的数量和分析的统计能力。一般来说，与传统流式细胞术相比，成像流式细胞术具有多种功能，例如同时分析细胞形态和表型、进行基于图像的分析、检测罕见事件以及验证流式细胞术数据13。这些优点增强了流式细胞术的能力，并有助于对细胞群进行更详细的检查。
这项研究为成像流式细胞术监测乳酸菌 （LAB） 中的自聚集提供了一种有价值的方案。这些革兰氏阳性杆菌是兼性厌氧菌，属于乳酸菌组。这些高效的葡萄糖发酵罐产生乳酸作为碳水化合物代谢的主要最终产物14.这些细菌是微生物组的有益核心成员，天然存在于人类和动物的胃肠道 （GIT） 以及女性的泌尿生殖道中15。因此，对其自聚集特性的准确表征具有高度的生物技术和临床意义。
我们之前的发现表明，不同益生菌菌株之间的自聚集基础水平不同。这种异质性受到用作碳源的不同碳水化合物的影响5.为了克服益生菌的这一基本特性，使用IFC监测饮食中碳水化合物对单细胞水平自聚集的影响。这种基于 IFC 的方法将传统流式细胞仪的功率和速度与显微镜的分辨率相结合。因此，它允许以表型定义的方式进行高速率复杂的形态测量16,17。这种方法可以扩展到其他益生菌和致病菌，结合荧光报告基因来监测基因表达，并结合荧光标记菌株来监测异质聚集体中特定细菌物种的存在和丰度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>14 mL culture tubes</td>
			<td>Falcon</td>
			<td>352051</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Baeton,Dickinson and Company</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Typtic Soy Broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Raffinose pentahydrate</td>
			<td>Sigma</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Lactobacilli MRS broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>288130</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-LOCK MICROPR. 1.5 mL (Eppendorf)</td>
			<td>FL medical</td>
			<td>23053</td>
			<td></td>
		</tr>
		<tr>
			<td>IDEAS Software</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150212_144049?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&#38;bd=1</td>
		</tr>
		<tr>
			<td>ImageStream X Mark II</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150121_205948?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr>
			<td>MOPS, 3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher bioreagents</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging flow cytometry to study microbial autoaggregation

有益菌和益生菌在其宿主中发挥着至关重要的作用，提供各种健康益处，包括对传染病的免疫力。乳酸杆菌科由革兰氏阳性菌组成，具有已证实的益生菌特性。本研究以乳酸杆菌科物种为模型，证明了单细胞高通量分析在研究细胞聚集方面的有效性。重点是分析这些有益物种对饮食中简单碳水化合物的反应。
该研究展示了成像流式细胞术 （IFC） 如何克服益生菌在存在和不存在碳水化合物的情况下组装的根本差异。IFC 将传统流式细胞术的功率和速度与显微镜的空间分辨率相结合，能够在有益细菌菌株和条件库中以表型定义的方式进行高速率复杂形态测量。该协议提供了对乳酸杆菌科物种自动聚集的见解，并揭示了它们对膳食碳水化合物的反应，有助于理解这些益生菌有益作用背后的机制。

结果表明，该方法可以很容易地测量乳酸菌中膳食糖分自聚集的差异。通过将个体与聚集体分离，该方法允许计算聚集事件的群体占所有事件的百分比，以响应饮食中的可发酵或不可发酵糖。此外，还可以测量处理之间总人口的平均大小是否存在差异。
图 3 中的代表性图像展示了每种 LAB 细菌的门控策略。在 LGG 和副干酪乳杆菌中，检测到单细胞?...

Read this Scientific Article Protocol about Author Spotlight: Advancing Research in Microbial Auto Aggregation Using Imaging Flow Cytometry at JoVE.com

作者聚焦：使用成像流式细胞术推进微生物自聚集的研究

该协议描述了一种使用成像流式细胞术测量微生物自聚集的定量方法。

成像流式细胞术研究微生物自聚集

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

本研究以乳酸菌属为模型，证明了成像流式细胞术分析在研究微生物自聚集方面的有效性。重点是分析乳酸菌物种的自聚集对饮食中简单碳水化合物的反应。测量自聚集最常用的方法是光谱光度法（测量微生物悬浮液、浊度或光密度）和传统显微镜技术（如明场、相衬和荧光显微镜）。在谈到高通量成像时，一个主要的实验挑战是单粒子分割。在此协议中，它是单个聚合。成像流式细胞术巧妙地解决了这一挑战，并为研究人员提供了研究自聚集的强大工具。

imaging-flow-cytometry-to-study-microbial-autoaggregation

Research

JoVE Journal

Biology

Imaging Flow Cytometry to Study Microbial Autoaggregation

590 次观看.  Ben-Gurion University of the Negev. 本研究以乳酸菌属为模型，证明了成像流式细胞术分析在研究微生物自聚集方面的有效性。重点是分析乳酸菌物种的自聚集对饮食中简单碳水化合物的反应。测量自聚集最常用的方法是光谱光度法（测量微生物悬浮液、浊度或光密度）和传统显微镜技术（如明场、相衬和荧光显微镜）。在谈到高通量成像时，一个主要的实验挑战是单粒子分割。在此协议中，它是单个聚?...

视频: 作者聚焦：使用成像流式细胞术推进微生物自聚集的研究

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

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 ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

测量细胞内Ca<sup&gt; +</sup在人类精子的变化采用4项技术:传统的荧光，停流荧光，流式细胞术及单细胞成像

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

一个小鼠红细胞亚群富集去核活动由多光谱成像流式细胞仪鉴定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析:一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...