This study investigates the role of Wnt5A in enhancing bacterial uptake in murine macrophages. By treating cells with Wnt5A, researchers observed improved phagosome maturation and bacterial degradation compared to control cells.
Take an adherent culture of murine macrophages.
Treat the test well with a buffer containing Wnt5A, a signaling molecule, and the control with buffer only.
Infect with a bacterial pathogen and incubate.
In the test cells, Wnt5A activates a signaling cascade that enhances cytoskeletal reorganization to boost bacterial uptake into phagosomes, whereas control cells lacking this signaling internalize fewer bacteria.
Wash to remove non-internalized bacteria, then incubate with a medium containing antibiotics to eliminate remaining extracellular bacteria.
Wnt5A signaling prepares the bacteria-containing phagosomes for fusion with lysosomes, which allows bacterial degradation.
Some bacteria escape by rupturing phagolysosomes.
In the control cells, limited phagosome maturation leads to increased escape.
Harvest the cells at defined timepoints. Add distilled water to induce osmotic shock, lysing the cells and releasing intracellular bacteria.
Spread the lysates on agar plates and incubate.
Count colonies to assess the greater time-dependent bacterial decline in Wnt5A-treated cells.
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