研究目的
To develop and describe a fluorescence-based proliferation assay for accurately identifying replicating bacteria within host cells at the subcellular level, using Staphylococcus aureus and macrophages as models, and to demonstrate its compatibility with additional cellular probes and applicability to both Gram-positive and Gram-negative bacteria.
研究成果
The fluorescence-based proliferation assay effectively identifies replicating bacteria within host cells and is compatible with additional cellular probes for niche characterization. It is applicable to both Gram-positive and Gram-negative bacteria, providing a valuable tool for studying host-pathogen interactions, though it requires careful optimization and is limited to microscopic analysis.
研究不足
The assay is best suited for fluorescence microscopy and not flow cytometry due to heterogeneity in phagosome contents within individual cells. Optimization is required for different bacterial species and strains, and care must be taken with probes like LysotrackerTM to avoid fixation artifacts.
1：Experimental Design and Method Selection:
The assay is based on labeling bacteria with a fluorescent proliferation dye (eFluorTM-670) that dilutes upon replication, allowing differentiation between replicating and non-replicating bacteria. It involves infection of macrophages with labeled bacteria, followed by fluorescence microscopy to detect dye dilution.
2：Sample Selection and Data Sources:
Bacterial strains include Staphylococcus aureus USA300, Yersinia pseudotuberculosis, Citrobacter rodentium, and Escherichia coli. Macrophage types include immortalized cell lines (e.g., RAW 264.7), primary murine bone marrow-derived macrophages, and primary human macrophages.
3：7), primary murine bone marrow-derived macrophages, and primary human macrophages.
List of Experimental Equipment and Materials:
3. List of Experimental Equipment and Materials: Fluorescent proliferation dye (eBioscienceTM Cell Proliferation Dye eFluorTM 670), GFP-expression vectors, tissue culture plates, coverslips, microscopes (e.g., Leica DMI6000 B), and various reagents for cell culture and staining.
4：Experimental Procedures and Operational Workflow:
Bacteria are labeled with eFluorTM-670, infected into macrophages at specific MOIs, incubated, and then analyzed by fluorescence microscopy. Additional stains like LysotrackerTM and LAMP-1 immunofluorescence are used for subcellular characterization.
5：Data Analysis Methods:
Images are acquired and processed using software like Leica LAS X and FIJI, with fluorescence intensity measurements and background subtraction to identify replicating bacteria.
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