Name: improvement of bacillus subtilis spore enumeration and label analysis in flow cytometry
Uploaded: 2023-06-30T12:40:00
Description: Watch this Scientific Journal Video about Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry at JoVE.com

这项研究的部分资金来自Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil （CAPES）-Finance Code 001;Governo do Estado do Amazonas，资源来自Fundação de Amparo à Pesquisa do Estado do Amazonas-FAPEAM;Conselho Nacional de Desenvolvimento Científico e Tecnológico （CNPq）.作者感谢健康工具技术发展计划PDTIS-FIOCRUZ使用其设施。

有关本协议中使用的所有材料、仪器和软件的详细信息，请参阅 材料表 。
1. 流式细胞术设置
<ol><li>与计算机耦合的流式细胞仪光学参数的对准<ol><li>登录到流式细胞仪软件。</li>
		<li>在软件工作区中，选择 “流式细胞仪”|”启动 并等待几分钟 |清洁模式 |静置液体。 注意：在样品采集之前，在细胞仪启动过程中去除气?...

<ol>
	<li>McKenney, P. T., Driks, A., Eichenberger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacillus+subtilis+endospore:+assembly+and+functions+of+the+multilayered+coat.">The Bacillus subtilis endospore: assembly and functions of the multilayered coat.</a> Nature Reviews. Microbiology. 11 (1), 33-44 (2013).</li><li>Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+probiotics.">Bacillus probiotics.</a> Food Microbiology. 28 (2), 214-220 (2011).</li><li>Ricca, E., Baccigalupi, L., Cangiano, G., De Felice, M., Isticato, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+vaccine+delivery+by+non-recombinant+spores+of+Bacillus+subtilis.">Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis.</a> Microbial Cell Factories. 13, 115 (2014).</li><li>Falahati-Pour, S. K., Lotfi, A. S., Ahmadian, G., Baghizadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+immobilization+of+recombinant+organophosphorus+hydrolase+on+spores+of+Bacillus+subtilis.">Covalent immobilization of recombinant organophosphorus hydrolase on spores of Bacillus subtilis.</a> Journal of Applied Microbiology. 118 (4), 976-988 (2015).</li><li>Harrold, Z., Hertel, M., Gorman-Lewis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Bacillus+subtilis+spore+isolation+and+quantifying+spore+harvest+purity.">Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity.</a> Journal of Microbiological Methods. 87 (3), 325-329 (2011).</li><li>Nicholson, W. L., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation,+germination+and+outgrowth.">Sporulation, germination and outgrowth.</a> Molecular biological methods for Bacillus. , (1990).</li><li>Mora-Uribe, P., 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=Characterization+of+the+adherence+of+Clostridium+difficile+spores:+the+integrity+of+the+outermost+layer+affects+adherence+properties+of+spores+of+the+epidemic+strain+R20291+to+components+of+the+intestinal+mucosa.">Characterization of the adherence of Clostridium difficile spores: the integrity of the outermost layer affects adherence properties of spores of the epidemic strain R20291 to components of the intestinal mucosa.</a> Frontiers in Cellular and Infection Microbiology. 6, 99 (2016).</li><li>Paidhungat, M., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ger+proteins+in+nutrient+and+nonnutrient+triggering+of+spore+germination+in+Bacillus+subtilis.">Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis.</a> Journal of Bacteriology. 182 (9), 2513-2519 (2000).</li><li>Schnizlein-Bick, C., Spritzler, J., Wilkening, C., Nicholson, J., O'Gorman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+TruCount+absolute-count+tubes+for+determining+CD4+and+CD8+cell+numbers+in+human+immunodeficiency+virus-positive+adults.+Site+Investigators+and+The+NIAID+DAIDS+New+Technologies+Evaluation+Group.">Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults. Site Investigators and The NIAID DAIDS New Technologies Evaluation Group.</a> Clinical and Diagnostic Laboratory Immunology. 7 (3), 336-343 (2000).</li><li>Godfrey, A., Alsharif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enumeration+of+viable+spores+by+flow+cytometry.">Rapid enumeration of viable spores by flow cytometry.</a> US Patent. , (2003).</li><li>Karava, M., Bracharz, F., Kabisch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+isolation+of+Bacillus+subtilis+spores+using+cell+sorting+and+automated+gating.">Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating.</a> PLoS ONE. 14 (7), e021989 (2019).</li><li>Genovese, M., Poulain, E., Doppler, F., Toussaint, R., Boyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+spore+enumeration+using+flow+cytometry:+A+proof+of+concept+for+probiotic+application.">Bacillus spore enumeration using flow cytometry: A proof of concept for probiotic application.</a> Journal of Microbiological Methods. 190, 106336 (2021).</li><li>Trunet, C., Ngo, H., Coroller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+permeabilization+and+activity+recovery+of+Bacillus+spores+in+adverse+conditions+for+growth.">Quantifying permeabilization and activity recovery of Bacillus spores in adverse conditions for growth.</a> Food Microbiology. 81, 115-120 (2019).</li><li>Tehri, N., Kumar, N., Raghu, H., Vashishth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+bacterial+spore+germination.">Biomarkers of bacterial spore germination.</a> Annals of Microbiology. 68, 513-523 (2018).</li><li>Isticato, R., Ricca, E., Baccigalupi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spore+adsorption+as+a+nonrecombinant+display+system+for+enzymes+and+antigens.">Spore adsorption as a nonrecombinant display system for enzymes and antigens.</a> Journal of Visualized Experiments. 145, e59102 (2019).</li><li>Song, 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=Killed+Bacillus+subtilis+spores+as+a+mucosal+adjuvant+for+an+H5N1+vaccine.">Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine.</a> Vaccine. 30 (22), 3266-3277 (2012).</li></ol>

传统方法，如菌落平板计数，不仅耗时，而且需要活细胞，并且不允许对灭活孢子进行定量5。Petroff-Hausser 室是一种替代方法，但它需要经验丰富的显微镜学家来执行。流式细胞术已被证明是实现此目的的有用替代方法。
Genovese等人12 描述了使用流式细胞术对 芽孢杆菌 的活细胞和孢子进行定量分析，使用两种核酸标记物和所用设备?...

枯草芽孢杆菌是一种杆状革兰氏阳性细菌，当环境条件不允许细胞生长时，能够产生静止孢子1。孢子是极其稳定的细胞形式，包括枯草芽孢杆菌在内的几种物种的孢子被广泛用作人类和动物使用的益生菌2。由于其抗性和安全性，枯草芽孢杆菌的孢子显示异源蛋白，已被提议作为粘膜佐剂、疫苗递送系统和酶固定平台 3,4。
为了从枯草芽孢杆菌中获得孢子，有必要使用特殊的培养基将其暴露在营养剥夺中。在获得并纯化这些孢子后，必须对其进行定量以提高测试效率 5,6。因此，应用某些方法来分析所获得的孢子的浓度。可以使用平板计数和 Petroff-Hausser 室（也称为计数室）。后者最初是为了确定血细胞的浓度而开发的;但是，可以在微生物学领域将其用于孢子计数7,8。尽管是用于细胞计数的标准方法，但读取是费力的，因为这种方法完全是手动的，其准确性取决于操作员的经验。
基于流式细胞术 （FC） 的分析先前已被提议作为检测 芽孢杆菌属标记细胞的快速、可靠和特异性方法。使用流式细胞术计数珠保证了常规检查（CD4 和 CD8 T 淋巴细胞的绝对计数）中细胞计数的可重复性，以及涉及能够使用流式细胞术检测和计数的颗粒的研究的发展9。Godjafrey 和 Alsharif 建议使用计数珠子对未标记的孢子进行 FC 定量10。描述了使用流式细胞术监测 芽孢杆菌属 的孢子形成。通过标记孢子DNA 10、11、12、13。另一项研究使用 FC 来评估孢子表面荧光标记蛋白质的数量15。
本研究试图使用商业计数微球来确保使用流式细胞术进行事件计数的可重复性标准。在此，我们建议在FC中使用计数珠进行细胞计数，以优化孢子计数并评估荧光标记抗体在孢子表面的偶联效率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC)</td>
			<td>Sigma</td>
			<td>341006</td>
			<td></td>
		</tr>
		<tr>
			<td>(N-hydroxysuccinimide) (NHS)</td>
			<td>Sigma</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human fluorescent antibody</td>
			<td>BioLegend</td>
			<td>501410</td>
			<td>APC anti-human IL-10</td>
		</tr>
		<tr>
			<td>Anti-mouse fluorescent antibody</td>
			<td>Thermo Scientific</td>
			<td>A32723</td>
			<td>Alexa Fluor Plus 488</td>
		</tr>
		<tr>
			<td>BD FACSCanto II</td>
			<td>&#160;BD</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>BD FACSDiva Cytometer Setup &#38; Tracking Beads Kit (use with BD FACSDiva software v 6.x)</td>
			<td>BD</td>
			<td>642412</td>
			<td>Quality control reagent</td>
		</tr>
		<tr>
			<td>BD FACSDiva Software v. 6.1.3</td>
			<td>BD</td>
			<td>643629</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Centrifuge MegaFuge 8R</td>
			<td>Thermo Scientific</td>
			<td>75007213</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Beads</td>
			<td>BD</td>
			<td>340334</td>
			<td>TruCount Tubes</td>
		</tr>
		<tr>
			<td>Eclipse 80i</td>
			<td>Nikon</td>
			<td></td>
			<td>Fluorescent Microsope</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Ludwig Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Microtubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Falcon</td>
			<td>352008</td>
			<td>5 mL polystyrene tube, 12 x 75 mm, without lid, non-sterile</td>
		</tr>
	</tbody>
</table>

improvement of bacillus subtilis spore enumeration and label analysis in flow cytometry

枯草芽孢杆菌的孢子已被提议用于不同的生物技术和免疫学应用;然而，越来越需要开发改进固定在孢子表面的抗原及其定量的方法。基于流式细胞术的分析以前已被提出作为检测枯草芽孢杆菌标记细胞的快速、可靠和特异性方法。在此，我们建议使用流式细胞术来评估荧光抗体（FA）在孢子表面的显示效率，并使用计数珠量化孢子的数量。
为此，我们使用溴化乙锭作为 DNA 标记物，并使用与孢子偶联的别藻蓝蛋白 （APC） 标记抗体作为表面标记物。孢子的定量是使用计数珠进行的，因为该技术在细胞检测方面表现出很高的准确性。使用流式细胞仪分析标记的孢子，证实了偶联。结果表明，DNA标记提高了流式细胞术定量的准确性，用于检测萌发的孢子。据观察，溴化乙锭不能标记休眠孢子;然而，该技术可以更精确地测定荧光蛋白与其表面偶联的孢子数量，从而有助于开展专注于在不同应用中使用孢子作为生物技术平台的研究。

在高压灭菌孢子（AS）样品中，分别使用计数珠和Petroff-Hausser方法检测2×103 个孢子/μl和1×103 个孢子/μl（图2）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65141/65141fig01.jpg"> 图1：孢子定量的一般方案。（A）用EtBr标记的孢子和（B）双标记的孢子。缩写?...

Watch this Scientific Journal Video about Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry at JoVE.com

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

该协议侧重于使用流式细胞术和计数珠来定量用溴化乙锭标记的细菌孢子。该方法还可用于分析完整孢子表面蛋白质的共价偶联。

枯草芽孢杆菌孢子计数和标记分析在流式细胞术中的改进

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

使用传统方法对枯草芽孢杆菌孢子进行细胞计数可能是一项艰巨的任务，因为这些方法可以完全手动进行，其准确性取决于操作员的经验。该协议使休眠和萌发孢子的计数成为可能。此外，该技术还可以确定荧光蛋白在其表面的应对百分比。鉴于该协议中存在的设置步骤，该愿景展示了该执行中的设施和技术理解以及护理细节。首先登录流式细胞仪软件。在软件工作区中，选择“流式细胞仪”，然后选择“流体启动”。然后选择“清洁模式”。最后，启动 SIT Flush。取 50 微升高压灭菌器孢子，用溴化乙锭以 0.05% 体积的稀释因子孵育 30 分钟，避光保存。用PBS洗涤孢子三次，以17,949G离心10分钟，然后重新悬浮在PBS中。接下来，按照制造商的稀释建议加入 10 微升珠子，并使用流式细胞仪分析样品。使用阴性对照作为参考，根据颗粒的形态测量和荧光特性定义门控策略。然后轻轻混合试管。将其连接到流式细胞仪探头，然后单击获取。要设置激光功率，请转到流式细胞仪窗口中的参数选项卡，将前向散射调整为 375，将侧向散射调整为 275。然后选择阈值选项卡并将其设置为 500。接下来，分析仅含有孢子的无染料样品以消除自发荧光。在参数选项卡中，将滤波器检测器三的电压调整为 603，将滤波器检测器五的电压调整为 538，以使用对照中的荧光区分阴性和阳性群体。然后单击“补偿”，并将滤波检测器 5 x 3 设置偏移量设置为 1。若要配置设备进行采集，请选择“实验”，选择实验布局，并将采集设置为 30， 000 个事件。调整参数后，在流式细胞仪中获取标记并包含珠子的样品的数据。将50微升孢子以17,949G离心10分钟。接下来，使用 25 微升 1-乙基-3-3-二甲氨基丙基碳二亚胺重新悬浮孢子并孵育 15 分钟。之后，向孢子悬浮液中加入浓度为 50 毫摩尔的 25 微升 N-羟基磺基琥珀酰亚胺。将孢子悬浮液在室温下孵育 30 分钟。如前所述，通过离心用PBS洗涤孢子三次。将荧光蛋白加入样品中，并在 15 摄氏度下孵育过夜。每毫升加入 10 毫克稀释至 1 至 50 的溴化乙锭，然后将样品放在冰上一小时，避光保存。如前所述，洗涤孢子并定义门后，在点图的 Y 轴上更改滤波器 3 的参数，并在点 图的 Y 轴上更改滤波器 5 的参数。计数微珠法在高压灭菌器孢子样品中检测到每微升 10 到第三个孢子的两次。高压灭菌孢子样品的溴化乙锭染色显示出比非高压灭菌孢子染色更高的平均荧光强度，表明遗传物质的染色更高。与非高压灭菌孢子相比，高压灭菌器孢子表面与APC标记的抗人白细胞介素10抗体偶联显示出更高的偶联效率。对溴化乙锭渗透的孢子的存在表明，在总种群中可能存在萌发的孢子。根据流式细胞术分析，观察到与萌发孢子相比，荧光抗体与休眠孢子的偶联百分比更高。此外，随着荧光抗体浓度的增加，观察到偶联孢子的百分比和平均荧光强度的增加。重要的是对电流磁珠进行彻底的均质化，以确保通过流式细胞术获得精确读数和准确计数孢子。标准化附着在孢子上的抗原浓度，加拿大研究分析了孢子作为疫苗抗体的使用。

Research

JoVE Journal

Biochemistry

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

Identification of Fatty Acids in Bacillus cereus

Identification of Fatty Acids in Bacillus cereus

脂肪酸鉴定<em&gt;蜡状芽孢杆菌</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

科研

生物化学

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

重组蛋白表达，结晶和生物物理研究<em&gt;芽孢杆菌</em&gt; - 保守的核苷酸焦磷酸化酶，BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

基于无标签阻抗的 GPCR 检测中用于提高吞吐量的逐步加量协议

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

使用定量流式细胞术分析荧光标记蛋白与人工磷脂微泡的相互作用

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...