Isolation of Mouse Bladder Epithelial Cells Containing Intracellular Bacterial Communities

Begin with bladder epithelial cells scraped from a mouse bladder infected with fluorescently labeled uropathogenic E. coli.

During infection, these bacteria invade epithelial cells and form intracellular bacterial communities, or IBCs—biofilm-like structures that protect them from host immune defenses.

Under a dissecting microscope, identify IBCs as large fluorescent aggregates confined within individual epithelial cells.

Assemble a mouth micropipetting setup using a pulled glass capillary connected to an aspirating pipette to isolate single IBC-containing epithelial cells.

Briefly dip the capillary tip into PBS to reduce capillary-driven fluid uptake.

Position the capillary near the selected fluorescent epithelial cell and apply gentle suction to draw in a single IBC-containing cell, avoiding uninfected cells and extracellular bacteria.

Expel the cell into a sterile microcentrifuge tube using slight positive pressure, preserving bacterial viability.

This method enables the isolation of epithelial cells harboring IBCs for single-cell analysis.

When all of the cells have been scraped, insert the unpulled end of a pulled glass capillary into the rubber plug of an aspirator tube and tightly f