免疫学研究
活细胞成像对
免疫系统中
健康和疾病的监测
免疫系统是一个高度复杂的器官、细胞和分子生物网络，可以保护宿主免受传染病甚至某些形式的癌症的侵害。免疫系统的内在复杂性及其参与众多基本生理过程，需要应用广泛的科学工具来深入了解免疫功能。免疫学领域严重依赖传统的体外技术，包括 ELISA、免疫印迹和流式细胞术。然而，这些方法提供的关于瞬时细胞间相互作用、免疫细胞定位及其形态的信息有限。
细胞培养的延时成像是一种强大的方法，它可以通过在生理相关条件下实时可视化复杂的生物过程来阐明免疫细胞行为的基本方面。活细胞成像不仅让科学家们能够解决未解决的研究问题，而且还能在这样做的同时节省时间和资源。
免疫细胞增殖的标记和无标记监测
检测免疫细胞的数量和活力
人外周血单核细胞 (PBMC) 是含有圆形细胞核的血细胞，如淋巴细胞、单核细胞和巨噬细胞。 作为免疫系统的主要组成部分，PBMC 在针对入侵病原体的细胞介导和体液免疫反应中发挥关键作用。 从全血中分离 PBMC 后，测量这些细胞的浓度，这可能是一个具有挑战性和耗时的过程。 CytoSMART Exact FL 荧光细胞计数器针对 PBMC 和其他难以检测的细胞类型进行了优化。 结合适当的荧光探针（例如 AO/PI），计数器可准确识别样品中的有核细胞，同时从计数中排除红细胞、血小板和其他细胞碎片。 此外，AO/PI 染色可用于评估 PBMCs 的生存能力。
Visualization of dynamic immune responses
吞噬作用是先天免疫反应的关键机制，中性粒细胞、巨噬细胞和其他专业吞噬细胞使用它从体内清除病原体和细胞碎片。 为了可视化和测量吞噬过程，使用 pH 敏感荧光探针标记外来物质，该探针在暴露于吞噬体的酸性环境中时会发出信号。 CytoSMART Lux3 FL 是一种便携式荧光活细胞成像仪，可以在最佳细胞培养条件下实时跟踪动态细胞过程，例如吞噬作用。 此外，CytoSMART Lux3 FL 的新对象计数算法可以自动计算图像中荧光对象的数量，并研究该数量随时间的变化情况。
免疫细胞趋化性的实时分析
某些免疫细胞（如中性粒细胞和 T 细胞）检测细胞外化学信号并响应它而迁移的能力在免疫系统功能和体内平衡中起着至关重要的作用。 细胞迁移分析需要应用高通量方法，这些方法可以可视化和量化细胞在空间和时间上的运动。 CytoSMART 活细胞成像平台，包括 CytoSMART Omni 和 Lux3 FL，可用于 单细胞（例如巨噬细胞、B 和 T 淋巴细胞）和 集体细胞迁移（例如肿瘤细胞）的直接可视化和跟踪。在单细胞迁移的情况下，可以使用市售（BioImaging Solutions, Inc. 的 MetaMorph）或免费（ImageJ 的 TrackMate）软件包分析记录的延时视频。 可以使用伤口愈合试验研究集体细胞迁移，通过集成的 CytoSMART 图像分析软件量化细胞运动的速率和效率的变化。
Videos
Appnotes
Cell Culture Monitoring - Lux2
Cell Motility Video Monitoring - Lux2
Cell Proliferation
In general monitoring cell growth is performed manually, by quickly investigating the confluency of the cell culture using a bright-field microscope. Investigation of confluency provides insight in the cell culture growth rate and cell viability.
The study performed here automated live-cell imaging is investigated as an alternative to manual inspection of confluency. A 96-well plate was seeded with rat brain glioma cells (C6) at varying densities and monitored over a 2-day period. The imaging process was performed inside a standard CO2-incubator for the entire experiment, ensuring optimal cell culture conditions.
Cell Culture Monitoring - Lux2
Setting up cell cultures is easy enough. However monitoring your cultures and optimization is time-consuming and cumbersome. Waiting for the ideal confluency, quickly studying effects of various media means taking your cells in and out of the incubator more often than you would like.
Visualizing cell cultures from inside an incubator using a compact microscope that facilitates live cell imaging can overcome these issues. While live cell imaging has been restricted to costly, high-end devices, the CytoSMART Lux2 offers an affordable and easy-to-use alternative for virtually any lab. The CytoSMART Lux2 can be set up in minutes, enabling untrained users to quickly perform their own time-lapse recordings.
Images and videos can be easily accessed and retrieved from the CytoSMART cloud portal. Advanced functions, such as reporting of cell confluency, cell migration analysis and the option to use automatic confluency email alerts, can be applied to inform the user when certain culture conditions are reached (for example, once the cell culture has reached the desired confluency). Hence, the CytoSMART Lux2 can be used in many different ways to facilitate cell culture work and research.
In the following appnote several examples of applications of the CytoSMART Lux2 will be shown.
- Culturing cells in hypoxic conditions
- Standardizing cell culturing conditions
- The effect of confluency on transfection efficiency
Cell Motility Video Monitoring - Lux2
Movement of cells plays a critical role in the development of cancer. Analyzing the motility of cells in vitro is therefor important for many cancer researchers.
Live-cell imaging, and in particular label-free live-cell imaging, is well suited to capture dynamic processes in cell culture without potential side-effects of markers or dyes on the cells. In the text below we will discuss the suitability of the CytoSMARTTM
Lux for several assays relevant for cancer research
We will discuss
- Introduction to the Lux cell monitoring system
- Tube formation assays
- 2D migration assays
- 3D invasion assays