研究目的
To outline a workflow for measuring single-cell survival of rat primary cortical neurons using longitudinal fluorescence microscopy to overcome limitations of standard cytotoxicity assays.
研究成果
The methodology enables continuous, single-cell monitoring of neuronal survival with high sensitivity, allowing for the identification of factors influencing cell death. It overcomes limitations of population-based assays and can be adapted for various experimental needs, providing a robust tool for studying neurodegeneration and other diseases.
研究不足
The method requires optimization for different cell types and substrates to prevent cell clumping or movement. Image processing may need manual adjustments for misalignments, and criteria for cell death scoring must be applied consistently. Transfection efficiency is low (5-10%), and the technique is limited to post-mitotic cells.
1：Experimental Design and Method Selection:
The method involves transient transfection of cells with fluorescent protein vectors, establishing a fiduciary for alignment, and imaging at regular intervals to track individual cells over time. Cell death is identified by changes in fluorescence, morphology, and fragmentation. Cox proportional hazards analysis is used for statistical comparison of survival rates.
2：Sample Selection and Data Sources:
Rat cortical neurons from embryonic day 19-20 pups are cultured on poly-D-lysine coated plates. Cells are transfected at DIV4 and imaged every 6-24 hours.
3：List of Experimental Equipment and Materials:
Includes fluorescent microscope with motorized stage, transfection reagents (e.g., Lipofectamine), plasmids encoding fluorescent proteins (e.g., mApple), 96-well plates, and software like FIJI and R for image processing and analysis.
4：Experimental Procedures and Operational Workflow:
Steps include material preparation, transfection, imaging with fiduciary alignment, image processing (stitching, stacking, background subtraction), scoring cell death based on predefined criteria, and performing Cox proportional hazards analysis using provided scripts.
5：Data Analysis Methods:
Data is analyzed using FIJI for image processing and R (specifically the survival package) for Cox proportional hazards analysis, log-rank tests, and visualization via Kaplan-Meier curves or cumulative risk plots.
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