研究目的
To describe the use of an automated optical system with eight parallel detectors for rapid and efficient data collection in FLIM-FRET imaging of Bcl-2 family protein interactions in a 384-well plate format.
研究成果
The study demonstrates rapid FLIM-FRET imaging of Bcl-2 family protein–protein interactions using an automated optical system with eight parallel detectors, enabling high-throughput screening in a 384-well format.
研究不足
The technical and application constraints include the time required for serial data acquisition and the potential for photobleaching and production of reactive oxygen species with increased laser power.
1：Experimental Design and Method Selection:
The study uses Fluorescence Lifetime Imaging Microscopy-Forster Resonance Energy Transfer (FLIM-FRET) to measure pairwise the direct interactions between Bcl-2 family proteins in live cells.
2：Sample Selection and Data Sources:
MCF7 human breast cancer cell line and BMKd3 cell line stably expressing mCerulean3-Bcl-XL were used.
3：List of Experimental Equipment and Materials:
Includes INO F-HS (FLIM-Hyperspectral) system, CellCarrier-384 Ultra microplate, and various cell culture components.
4：Experimental Procedures and Operational Workflow:
Detailed steps for cell culture, transfection, and FLIM-FRET imaging are provided.
5：Data Analysis Methods:
The data is analyzed using a fitting algorithm to estimate fluorescence lifetime and FRET efficiency.
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