This article details a method for isolating intracellular bacterial communities (IBCs) from bladder epithelial cells infected with uropathogenic E. coli. The technique allows for the analysis of single IBC-containing cells, providing insights into bacterial behavior within host cells.
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 fit the narrow end of a 1 milliliter pipette tip into the other open end of the tube.
Tightly fit the narrow end of a 2 milliliter aspirating pipette into the open, wider end of the 1 milliliter pipette tip and place the scraped cell suspension under a dissecting microscope. Using a 20 to 40X magnification, identify the IBCs as large fluorescent aggregates and dip the fine end of the glass capillary into a fresh tube of PBS for 1 second to reduce the uptake of unwanted volume through capillary action.
When an IBC of interest has been located, slowly bring the open end of the capillary tube toward the IBC and apply a very small suction force to the aspirating pipette to guide the IBC into the glass capillary. Then, move the capillary to an empty 1.5 milliliter centrifuge tube and apply a slight positive pressure to expel the droplet and the IBC into the tube.
繁體中文
