研究目的
To present a protocol for live cell imaging of migrating T-cells to understand their navigation and motility, which can offer new therapeutic opportunities.
研究成果
The protocol provides a reproducible method for real-time imaging of T-cell migration, enabling the investigation of dynamic behaviors such as speed and direction. It can be adapted for studying intracellular signaling events, with potential applications in therapeutic development.
研究不足
Live cell imaging is challenging due to potential cell damage from laser illumination, need for constant environmental conditions, and the non-adherent nature of T-cells. Cytotoxicity and photobleaching of dyes can affect results, and imaging parameters must be optimized to minimize damage.
1：Experimental Design and Method Selection:
The protocol involves live cell imaging using an automated microscope to track T-cell migration in real-time, with steps for plate coating, cell activation, staining, and imaging.
2：Sample Selection and Data Sources:
Human primary T-cells isolated from blood samples are used.
3：List of Experimental Equipment and Materials:
Includes RPMI1640 medium, T-cells, PBS, coating reagents, LFA-1 activation buffer, 96-well glass bottom plates, drugs like Nocodazole, staining dyes (CellMask?, Hoechst 33342), automated microscope (IN CELL Analyzer 2200), and image analysis software (Imaris v
4：0). Experimental Procedures and Operational Workflow:
Steps include plate coating with rICAM-1, T-cell activation and staining, live cell imaging with specific microscope settings, and image analysis using tracking algorithms.
5：Data Analysis Methods:
Image analysis is performed using Imaris software to track cells and plot migration parameters such as speed, displacement, and persistence.
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