荧光激活细胞分拣，用于分离瘦杆菌细胞群
2020年5月31日
珊瑚创造了对人类和海洋生物都重要的生物多样性生态系统。然而，我们仍然不明白许多珊瑚细胞的全部潜力和功能。在这里，我们提出了为石珊瑚细胞群的分离、标记和分离而开发的协议。
该协议有助于在以前不可能确定的描述水平上识别和隔离活珊瑚细胞种群。这种技术的主要优点是，它允许访问一个细胞特异性水平，到目前为止，已经基本上无法实现。展示这个程序的将是山农·赛格博士，一个研究助理，来自西尔维斯特综合癌症中心的流细胞学共享资源。
要设置活珊瑚细胞分拣的流动细胞仪，请在流式细胞仪软件中打开一个新的项目模板，并在激光面板中选择合适的激光器进行分析。然后，在适当的图中创建侧散点图与前进散点图，以设置浇注和分析策略。对于活珊瑚细胞的流动细胞测量分析和分拣，将控制未污染的细胞样本加载到细胞仪的样品室中，然后开始读取过程。
由于珊瑚细胞特别脆弱，请保持细胞计的低气压，以防止细胞解。在散点图中，调整光倍增器电压以居中点并开始记录事件。在前进与侧散射图中，在向前散射 x 轴上 10 到 4 个标记周围的单元格周围创建一个栅极。
用染色细胞的第一个样本替换控制样本，并在暂停前记录大约 10，000 个细胞。然后对染色样本组唯一的事件进行门控。要对活珊瑚细胞进行排序，在选择流式细胞仪软件中感兴趣的细胞群后，将一个包含250至500微升适当收集溶液的微离心管放入细胞计收集室，供每个细胞量采集室进行排序。
一旦经过适当的时间来清除碎片，开始整理所需的种群，收集从1万到数百万个细胞的任何地方。每次使用新的污渍、物种或分拣器时，将至少2万个感兴趣的细胞分类成500微升染色介质，以便对感兴趣的群体进行纯度检查，并重新分析分拣细胞，以确认事件正在用于初始分拣的大门内读取。对于体外研究，将分类的细胞储存在冰上，直到它们被转移到培养箱或灭菌环境。
通过在向前和侧面散射图上创建栅极选择，引入与珊瑚细胞不具有相同形状或粒度的其他非细胞粒子。通过使用直方图比较未染色和染色细胞悬浮液的 DAPI 信号，并找出高 DAPI 表达细胞，可以排除死细胞。同时，通过对远红色通道中信号高的细胞进行浇注，可以去除自荧光共生细胞。
经过这个迭代的浇注过程，剩余的细胞代表缺乏共生亚的完整活珊瑚细胞种群。然后，这些细胞可以通过染色（如活性氧物种活性和/或糖体含量）的染色，分类为不同的细胞子组。请记住，某些细胞比另一些细胞更脆弱，并尽可能小心地运行此过程。
一旦分离出特定的细胞，它们就可用于x-vivo功能测定，如建立细胞培养和创建转录体配置文件。
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概述
本研究针对需要更好地理解珊瑚细胞的需求，珊瑚细胞对海洋生态系统的生物多样性至关重要。作者提出了一种新的石珊瑚细胞种群分离和标记的协议，实现了前所未有的细胞特异性水平。
关键研究组成部分
研究领域
- 珊瑚生物学
- 细胞分离技术
- 生态系统生物多样性
背景
- 珊瑚为海洋生物形成了重要的生态系统。
- 目前对珊瑚细胞功能的了解有限。
- 分离特定珊瑚细胞种群对进一步研究至关重要。
使用的方法
- 流式细胞术用于活细胞分选
- 石珊瑚作为模型生物
- 用于识别和分离特定细胞类型的门控策略
主要结果
- 该协议允许识别和分选活珊瑚细胞种群。
- 确保分选细胞的纯度和活力，以用于未来的测定。
- 促进了对亚群体及其功能的研究。
结论
- 该研究提出了一种可靠的珊瑚细胞分离方法，增强了研究潜力。
- 研究结果为珊瑚生物学和生态系统健康提供了宝贵的见解。
常见问题
Executive Industry Relevance
Isolating specific cell populations from complex tissues remains a critical challenge in early-stage target validation and mechanistic de-risking. Fluorescence-activated cell sorting (FACS) enables high-resolution cellular interrogation, supporting predictive confidence in pathway-specific responses. This capability is directly applicable to biopharma discovery workflows requiring precise functional assays and transcriptomic profiling of rare or fragile cell types.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses at single-cell resolution by isolating live populations for functional validation.
- Operational Value: Reduces mechanistic ambiguity by separating viable cell subpopulations from debris, dead cells, and symbiont-associated signals.
- Predictive Value: Supports portfolio triage by generating purified inputs for downstream assays that clarify target engagement and pathway modulation.
Screening & Assay Development
- Scientific Value: Produces standardized, viable cell suspensions suitable for reproducible functional assays and dose-response profiling.
- Operational Value: Establishes gating strategies that ensure assay readiness and minimize variability across experimental runs.
- Scalability: Allows collection of sufficient cell numbers (10,000 to several million) for multi-well screening and omics applications.
Translational & Preclinical Research
- Translational Continuity: Bridges discovery and preclinical stages by enabling consistent isolation of disease-relevant cell populations for mechanistic follow-up.
- Biomarker Alignment: Facilitates staining for stress-response markers (e.g., reactive oxygen species, lysosome content) to correlate cellular phenotypes with functional outcomes.
- Risk-Adjusted Advancement: Purified populations improve data reliability in preclinical models, reducing false positives from heterogeneous samples.
Pipeline & Workflow Integration
FACS integrates into the discovery continuum from target hypothesis testing through lead identification to preclinical validation by enabling precise cellular inputs at each stage.
- Discovery Biology: Supports hypothesis testing by isolating live coral (or analogous mammalian) cell populations for pathway-specific functional interrogation.
- Screening: Delivers standardized, gated cell populations that enhance assay reproducibility and quantitative readout reliability.
- Analytics: Generates high-purity samples enabling accurate transcriptomic, proteomic, or metabolic profiling to compare experimental conditions.
- Translational Research: Connects early discovery to preclinical validation through consistent preparation of viable, stress-responsive cell subsets.
- Enterprise Reuse: Functions as a reusable platform capability across projects requiring subcellular resolution, independent of model system.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing noise from non-target cells and enabling mechanistic de-risking of targets.
- Operational Value: Standardizes sample preparation through reproducible gating, low-pressure sorting, and viability-preserving collection methods.
- Strategic Value: Improves go/no-go decisions by providing cleaner biological data, reducing late-stage failure risk from flawed target assumptions.
- Portfolio Impact: Enables risk-adjusted prioritization through reliable subpopulation-specific data that informs advancement criteria.
Implementation Considerations
- Requires expertise in flow cytometry setup, including laser selection, voltage optimization, and scatter plot gating for fragile cells.
- Dependent on instrumentation capable of low-pressure sorting and multi-parameter detection (forward/side scatter, fluorescence channels).
- Necessitates cross-team standardization of staining protocols, collection media, and gating thresholds to ensure reproducibility.
- Must account for variability in cell fragility across species or conditions, requiring iterative optimization of shear stress and staining duration.
- Limited by sample preparation quality; enzymatic or mechanical dissociation must preserve epitope integrity and cell viability for accurate sorting.
