Culture A549 epithelial cells as described in the text protocol. After one wash and trypsinization of the cells at 37 degrees Celsius, dilute the cell suspension to 5,000 cells per milliliter using complete medium containing 5 micrograms per milliliter of gentamicin.
Prior to bacterial infection, replace the medium in the wells with F12 medium and incubate overnight at 37 degrees Celsius. Wash the cell monolayer three times with 1 milliliter of PBS at room temperature. Then, place the plate on ice and add 200 microliters of pre-chilled F12 medium, containing 10 micrograms per milliliter of vitronectin.
After incubating the plate at 4 degrees Celsius for 1 hour, discard the solution by pipetting, and wash the cell layer twice with 1 milliliter of PBS at room temperature. Add 100 microliters of freshly grown Hif wild-type in F12 medium to each well. Incubate the plate for 2 hours at 37 degrees Celsius. Then aspirate the medium with a pipette, and wash the A549 epithelial cells three times with PBS at 50 microliters per well of cell detachment solution and follow with a 5-minute incubation at 37 degrees Celsius.
Next, add 50 microliters of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells from each well to a 6-milliliter glass tube containing 4 glass beads. Lyse the cells at room temperature by vortexing for 2 minutes.
After diluting the cell lysate 100-fold, plate 10 microliters of the diluted sample onto a chocolate agar plate. Count the colonies following an overnight incubation at 37 degrees Celsius.
