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

<ol>
	<li>Trunk, T., Khalil, H. S., Leo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+autoaggregation.">Bacterial autoaggregation.</a> AIMS Microbiology. 4 (1), 140-164 (2018).</li><li>Ishikawa, M., Nakatani, H., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtaA,+a+new+member+of+the+trimeric+autotransporter+adhesins+from+Acinetobacter+sp.+Tol+5+mediating+high+adhesiveness+to+various+abiotic+surfaces.">AtaA, a new member of the trimeric autotransporter adhesins from Acinetobacter sp. Tol 5 mediating high adhesiveness to various abiotic surfaces.</a> PLoS One. 7, e48830 (2012).</li><li>Inoue, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+low-molecular-weight+component+protein,+Flp,+in+Actinobacillus+actinomycetemcomitans+fimbriae.">Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae.</a> Medical Microbiology and Immunology. 42 (4), 253-258 (1998).</li><li>Boddey, J. A., Flegg, C. P., Day, C. J., Beacham, I. R., Peak, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-regulated+microcolony+formation+by+Burkholderia+pseudomallei+requires+pilA+and+enhances+association+with+cultured+human+cells.">Temperature-regulated microcolony formation by Burkholderia pseudomallei requires pilA and enhances association with cultured human cells.</a> Infection and Immunity. 74 (9), 5374-5381 (2006).</li><li>Suissa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+differences+in+the+functional+responses+of+Lactobacillaceae+strains+to+fermentable+sugars.">Context-dependent differences in the functional responses of Lactobacillaceae strains to fermentable sugars.</a> Frontiers in Microbiology. 13, 949932 (2022).</li><li>Isenring, J., Geirnaert, A., Lacroix, C., Stevens, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistable+auto-aggregation+phenotype+in+Lactiplantibacillus+plantarum+emerges+after+cultivation+in+in+vitro+colonic+microbiota.">Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota.</a> BMC Microbiology. 21, 268 (2021).</li><li>Kos, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+and+aggregation+ability+of+probiotic+strain+Lactobacillus+acidophilus+M92.">Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92.</a> Journal of Applied Microbiology. 94 (6), 981-987 (2003).</li><li>Zommiti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assessment+of+the+probiotic+properties+and+bacteriocinogenic+potential+of+Pediococcus+pentosaceus+MZF16+isolated+from+artisanal+tunisian+meat+&amp;#34;Dried+Ossban&amp;#34;.">In vitro assessment of the probiotic properties and bacteriocinogenic potential of Pediococcus pentosaceus MZF16 isolated from artisanal tunisian meat &amp;#34;Dried Ossban&amp;#34;.</a> Frontiers in Microbiology. 9, 2607 (2018).</li><li>Suissa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+rewiring+of+the+probiotic+bacterium+rhamnosus&lt;/em&gt;+GG+contributes+to+cell-wall+remodeling+and+antimicrobials+production.">Metabolic rewiring of the probiotic bacterium rhamnosus&lt;/em&gt; GG contributes to cell-wall remodeling and antimicrobials production.</a> bioRxiv. , (2023).</li><li>Connell, J. L., Kim, J., Shear, J. B., Bard, A. J., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+quorum+sensing+in+3D-printed+bacterial+aggregates+using+scanning+electrochemical+microscopy.">Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (51), 18255-18260 (2014).</li><li>Misawa, N., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+autoagglutination+activity+by+Campylobacter+jejuni.">Detection and characterization of autoagglutination activity by Campylobacter jejuni.</a> Infection and Immunity. 68 (11), 6168-6175 (2000).</li><li>Sherlock, O., Schembri, M. A., Reisner, A., Klemm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+roles+for+the+AIDA+adhesin+from+diarrheagenic+Escherichia+coli:+cell+aggregation+and+biofilm+formation.">Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation.</a> Journal of Bacteriology. 186 (23), 8058-8065 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry.">Imaging flow cytometry.</a> Nature Reviews Methods Primers. 2, 87 (2022).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+characteristics+of+lactic+acid+bacteria+and+the+expanding+applications+in+food+industry.">Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry.</a> Frontiers in Bioengineering and Biotechnology. 9, 612285 (2021).</li><li>Turroni, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dialogue+between+the+human+gut+microbiota+and+the+host:+a+Lactobacillus+and+Bifidobacterium+perspective.">Molecular dialogue between the human gut microbiota and the host: a Lactobacillus and Bifidobacterium perspective.</a> Cellular and Molecular Life Sciences. 71, 183-203 (2014).</li><li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochemica et Cytobiologica. 45, 279-290 (2007).</li><li>Dashkova, V., Malashenkov, D., Poulton, N., Vorobjev, I., Barteneva, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+for+phytoplankton+analysis.">Imaging flow cytometry for phytoplankton analysis.</a> Methods. 112, 188-200 (2017).</li><li>Maan, H., Gilhar, O., Porat, Z., Kolodkin-Gal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+colonization+of+arabidopsis+thaliana+roots+induces+multiple+biosynthetic+clusters+for+antibiotic+production.">Bacillus subtilis colonization of arabidopsis thaliana roots induces multiple biosynthetic clusters for antibiotic production.</a> Frontiers in Cellular and Infection Microbiology. 11, 722778 (2021).</li><li>Maan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+reveals+a+dual+role+for+exopolysaccharides+in+biofilms:+To+promote+self-adhesion+while+repelling+non-self-community+members.">Imaging flow cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members.</a> Computational and Structural Biotechnology Journal. 20, 15-25 (2022).</li><li>Konieczny, M., Rhein, P., Czaczyk, K., Bialas, W., Juzwa, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+to+study+biofilm-associated+microbial+aggregates.">Imaging flow cytometry to study biofilm-associated microbial aggregates.</a> Molecules. 26 (23), 7096 (2021).</li><li>Niederdorfer, R., Peter, H., Battin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attached+biofilms+and+suspended+aggregates+are+distinct+microbial+lifestyles+emanating+from+differing+hydraulics.">Attached biofilms and suspended aggregates are distinct microbial lifestyles emanating from differing hydraulics.</a> Nature Microbiology. 1, 16178 (2016).</li></ol>

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于数值图像的特征，将传统流式细胞术中大样本量的统计显着性与标准显微镜的每细胞信息含量相结合16。
以前，IFC 已被用于研究各种细菌行为，例如枯草芽孢杆菌中的抗生素产生和双物种生物膜的组成18,19。虽然有零星使用 Imagestream 流式细胞术测量自聚集的报道20，但它被认为是其在研究益生菌5 中的有用性的首次证明。研究结果表明，IFC是高效和可靠的，在研究LAB菌株的微生物聚集方面，IFC可以替代传统的流式细胞术和显微镜法，特别是考虑到菌株之间自聚集的基础水平的显着差异5。这种方法对于多种菌株和生长条件的准确高通量筛选特别有价值。
但是，基于 IFC 的方法具有一定的局限性和注意事项。与传统的显微镜方法相比，它需要更多的单元格来生成数据集。此外，由于该方法的固有特性，IFC图像的光学和数字分辨率相对低于传统显微镜。虽然传统的流式细胞术缺乏光学扫描的可靠性和质量保证，但 Imagestream 流式细胞术通常用作传统流式细胞术的替代品，而不是荧光显微镜。此外，确定区分单个细胞和聚集体的面积值是一个关键步骤，其准确识别对于避免丢失重要数据至关重要。如方案中所述，该值因细菌种类而异，应仔细确定。
可以进行进一步的发展以扩大该方法的应用，例如研究其他环境因素下的聚集。将图像分析与流式细胞术数据相结合，可以更全面地表征微生物聚集体，包括大小、形状和组成。该方法还可用于增强自然发生的水生栖息地中微生物聚集体的高通量表征21。此外，通过标记靶物种和细胞类型，它可以很容易地适应于检查复杂微生物组群落中的聚集。
总体而言，研究结果强调，这种方法简单明了，信息丰富，对于评估单一物种和复杂群落中细菌的聚集特性非常有用。它通过比较不同缺失、过表达菌株以及各种化学和物理操作之间的细胞数量和聚集体大小，可以更好地理解自聚集的分子机制。

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><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>&amp;nbsp;Details available at: https://www.merckmillipore.com/INTL/en/20150212_144049?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&amp;amp;bd=1</td></tr><tr><td>ImageStream X Mark II</td><td>Amnis/EMD Millipore</td><td>N/A</td><td>&amp;nbsp;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、小聚集体、大聚集体和链（图 3）。然而，链和较大的聚集体无法分开。
图 4 中的代表性图像揭示了不同 LAB 菌株对可发酵或不可发酵糖的聚集特性存在很大差异，这些差异通过群体百分比和聚集事件的平均大小来衡量5。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65788/65788fig01.jpg"> 图 1：样品制备过程的工作流程。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65788/65788fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65788/65788fig02.jpg"> 图 2：用于评估聚合事件群体和大小的门控策略。 （A） 使用明场通道基于梯度 RMS 对所有事件的聚焦事件进行门控。（B） 通过绘制明场通道的面积 （μm2） 与纵横比（宽度除以最佳拟合椭圆的长度）的关系，将单个聚集体和小聚集体与较大聚集体分离。（C） 聚集事件中明场通道的面积提供了有关粒子大小的信息。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65788/65788fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65788/65788fig03.jpg"> 图 3：设门策略的单元格图像。 （A） LGG。（B） TSB中的 副干酪乳杆菌 （对照）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65788/65788fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65788/65788fig04.jpg"> 图 4：不同条件下聚集事件群体的代表性明场图像。 （A） LGG。（B） 副干酪乳杆菌。（C） 植物乳杆菌。（D） 干酪乳杆菌。条件包括 TSB 培养基（对照）、补充葡萄糖和棉子糖 （1% w/v） 的 TSB 培养基和补充葡萄糖 （1% w/v） 和缓冲液的 TSB 培养基。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65788/65788fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充编码文件1： 以 LGG 模板为例的 .ast 文件。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65788/Supporting%20.ast%20file%20as%20example%20for%20LGG%20.zip" target="_blank">请点击这里下载此文件。</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

Imaging Flow Cytometry to Study Microbial Autoaggregation

608 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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

科研

生物工程

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

环境科学

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

将流体设备与显微镜和流式细胞学相结合，研究跨空间尺度的多孔介质中的微生物传输

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

免疫与感染

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

小鼠B细胞发育的流式细胞学表征

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

成像流式细胞术研究微生物自聚集

副本

摘要

探索更多视频

此视频中的章节