Guo Yuting, Baumgart Sabine, Stärk Hans-Joachim, Harms Hauke, Müller Susann
Department of Environmental Microbiology, Helmholtz Centre for Environmental ResearchLeipzig, Germany.
Department of Immune Monitoring, German Rheumatism Research Centre, An-Institute of the Leibniz AssociationBerlin, Germany.
Front Microbiol. 2017 Jul 17;8:1326. doi: 10.3389/fmicb.2017.01326. eCollection 2017.
Mass cytometry (Cytometry by Time of Flight, CyTOF) allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell. For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells' silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing cells as test organisms. A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected. An effective mass cytometry protocol was developed for the detection and quantification of silver in single bacterial cells of different physiological states. The silver quantities were generally heterogeneously distributed among cells in a population, the degree of which was dependent on micro-environmental conditions and on silver applied either in ion or nanoparticle-aggregated form.
质谱流式细胞术（飞行时间质谱细胞术，CyTOF）可基于特定的金属基细胞标志物对单细胞进行表征。此外，每个细胞还能检测到质谱范围内的其他金属，如银。已知细菌对银敏感，因此开发了一种方案来测量每个群体中受影响细胞的数量以及每个细胞中的银含量。对于质谱流式细胞术，钌红用作群体中所有细胞的标志物，同时顺铂的平行应用可区分活细胞和死细胞。通过质谱流式细胞术测量每个细胞的银含量以及群体中含银细胞的频率。此外，通过流式细胞术分析活/死亚群，并基于钌红和碘化丙啶双重染色通过细胞分选进行区分。通过使用电感耦合等离子体质谱（ICP-MS）结合细胞分选在整体水平上对细胞的银负载进行验证。该方案是通过将快速生长和非生长细胞作为测试生物体来开发的。开发了一种用于标记细菌以便通过质谱流式细胞术进行分析的工作流程。测试了三个不同参数：钌红提供群体中所有细菌细胞的计数，而连续应用的顺铂标记死细胞的频率。通过含银细胞的不同频率检测到明显的群体异质性。每个细胞的银含量也可很好地测量。一般来说，AgNP-10处理导致更高的死细胞频率、更高的含银细胞频率和更高的每个细胞的银含量。由于游离和结合银离子的假定化学平衡，活细胞和死细胞与银的结合量相等，且这一现象在指数生长期间更为明显。通过ICP-MS检测到每个细菌细胞中高达1.5 fg的银。开发了一种有效的质谱流式细胞术方案，用于检测和定量不同生理状态下单细菌细胞中的银。银含量通常在群体中的细胞间呈异质分布，其程度取决于微环境条件以及以离子或纳米颗粒聚集形式应用的银。
