Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells
Transcription
Begin with a suspension of actively growing Porphyromonas gingivalis, anaerobic pathogenic bacteria.
Add BCECF-AM, a non-fluorescent, cell-permeable dye.
Active cytoplasmic esterases hydrolyze the dye into a fluorescent, cell-impermeable form, staining the bacteria.
Centrifuge. Resuspend the bacteria in anaerobic media.
Add the labeled bacteria to multi-well plate wells containing coverslips with adhered human endothelial cells.
Incubate in an anaerobic chamber.
The bacterial fimbriae bind to the endothelial cell surface receptors, initiating intracellular signaling cascades.
This leads to actin rearrangement and bacterial internalization within phagosomes.
Post-incubation, wash coverslips with buffer to remove non-internalized bacteria.
Fix the cells with paraformaldehyde to preserve the cellular structures. Permeabilize the cells with a non-ionic detergent to access intracellular targets.
Add fluorescently labeled phalloidin, which binds to the actin filaments.
Place the coverslip over a mounting medium containing DAPI, which stains the DNA.
Observe the fluorescent Porphyromonas gingivalis within endothelial cells with fluorescent actin, confirming bacterial internalization.
