Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging

Overview

This video demonstrates the bacterial adherence assay to evaluate GFP-tagged bacterial adherence to epithelial cell culture to study the dynamics of host-pathogen interactions using fluorescence microscopy-based imaging.

Protocol

1. Bacterial adherence and host cell staining

1. First, wash the seeded A549 cell monolayers three times with warm 1x PBS. For each wash, add 100 μL of 1x PBS to each well, gently pipette up and down three times, dispose of 1x PBS or wait for 10 s after addition, and then vacuum to remove 1x PBS. To determine the kinetics of bacterial association, overlay the cells with 100 µL of desired concentrations of bacterial suspension with different MOIs (0, 1, 10, and 100). Spin down the bacteria at 200 x g for 10 min and incubate the infected A549 cells at 37 °C, 5% CO₂ for an additional 1 h.
   NOTE: In this experiment, each condition had a technical triplicate.
2. Remove the unbound bacteria by washing the monolayers five times with warm 1x PBS as described above. Add 100 µL of 4% formaldehyde (in 1x PBS) into each well of the 96-well plates to fix the cells. Let the plates sit on ice for 15 min and then wash off the fixation solution using 1x PBS three times.
3. To stain the nuclei, add 50 ng/mL of 4′,6-diamidino-2-phenylindole (DAPI) and incubate it for 10 min at RT. After incubation, wash the wells three times using 1x PBS. Cover the infected A549 cells with 100 µL of 1x PBS to avoid drying. Process for the next step or store the plate at 4 °C for up to 2 days in the dark.

Materials

Name	Company	Catalog Number	Comments
10x PBS	VWR	45001-130
4′,6-diamidino-2-phenylindole (DAPI)	Thermo Fisher	62248	Host cell staining dye
96-well plate	Corning	3882	Half area well, flat clear bottom
A549 cells	ATCC	CCL 185	Mammalian cell line
Centrifuge	Eppendorf	5810R
OD600 DiluPhotometer	IMPLEN
Typical soy broth	Growcells	MBPE-4040
F-12k medium	ATCC	302004	A549 cell culture medium
Fetal bovine serum	Corning	35-016-CV