Overview
In this video, we demonstrate an assay to study the role of vitronectin in Haemophilus influenzae adherence to the host epithelial cells.
Protocol
1. Study of Vn-dependent adherence of bacteria to epithelial cells
- Culture A549 cells (type II alveolar epithelial cells) in a 75-cm2 tissue culture flask with F12 medium supplemented with 10% (vol/vol) fetal calf serum (FCS) (complete medium) and 5 µg/mL gentamicin. Incubate the flask in an incubator with 5% CO2 at 37 °C for 3 days until 80% confluent (confluency can be estimated by visualizing surface coverage by the cells using an inverted microscope). The following four steps describe how to prepare the epithelial cells for the assay.
- Wash the cell monolayer in the flask twice with 20 mL of PBS by gentle swirling. To detach the cells from the flask surface, add 2 mL of cell detachment enzyme solution and incubate the flask for 5 min at 37 °C. Tap the flask with your palm to detach all the cells from the plastic surface. Pipette up and down a few times to disperse cell clumps.
- Add 18 mL of F12 complete medium to the flask and transfer the entire cell suspension to a 50-mL sterile Falcon tube. Centrifuge the cell suspension for 5 min at 200 x g at RT and discard the supernatant. Resuspend the cell pellet in 10 mL of F12 complete medium.
- Remove 10 µL of the cell suspension and place it in an Eppendorf tube, then add 90 µL of Trypan blue solution. Load the sample into a hemocytometer (depth 0.1 mm) after placing the coverslip.
- Count all viable cells in areas A, B, C, and D (each field consists of 16 squares, and each square has an area of 0.0025 mm2), then calculate the average number of cells ([A+B+C+D]/4). Calculate the number of cells per milliliter using the following equation:
Viable cells/mL = average cell count × 104 × dilution factor.
NOTE: Here, the dilution factor is 10. - Dilute the cell suspension to 5.0 x 103 cells/mL using a complete medium containing 5 µg/mL gentamicin. Dispense 500 μL of cell suspension into each well of a 24-well cell culture plate. Incubate the plate at 37 °C in 5% CO2 until the cells are 90% confluent.
- Prior to bacterial infection, remove the medium from the wells and add F12 medium (without FCS), then incubate overnight at 37 °C.
- Wash the cell monolayer three times with 500 µL of PBS at RT. Place the plate on ice and add 100 µL of pre-chilled F12 medium containing 10 µg/mL of Vn. Incubate plate at 4 °C for 1 h. For control wells, add only F12 medium.
- After incubation, discard the solution by pipetting and wash the cell layer twice with 1 mL of PBS at RT.
- Culture Hif clinical isolates in brain-heart infusion (BHI) medium supplemented with 10 µg/mL NAD and hemin at 37 °C with shaking at 200 rpm. Use Luria-Bertani medium to culture E. coli. Harvest Hif at mid-log phase (OD600 = 0.3) and resuspend the bacteria in phosphate-buffered saline (PBS), pH 7.2, containing 1% (w/v) bovine serum albumin (BSA) (blocking buffer; PBS-BSA). Adjust the suspension to 109 CFU/mL. Resuspend a culture of freshly grown Hif M10 in F12 medium (2×108 CFU/mL). Add 100 µL of this bacterial suspension to each well and incubate the plate for 2 h at 37 °C.
NOTE: Each well contains approximately 2 x 105 A549 cells. For infection, 2 x 107 bacterial CFU were added, corresponding to a multiplicity of infection of 100. - Remove the medium by pipetting and wash the A549 epithelial cells three times with PBS. Add 50 µL/well of cell detachment solution and incubate the plate for 5 min at 37 °C.
- Next, add 50 µL of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells (≈100 μL [i.e., the entire volume]) from each well to a 6-mL glass tube containing four glass beads. Lyse the cells at RT by vortexing for 2 min.
- Dilute 10 µL of the cell lysate 100-fold by adding 10 µL of lysate to 990 µL of F12 medium. Plate 10 µL of the diluted sample onto a chocolate agar plate.
- Incubate the chocolate agar plate at 37 °C overnight, then count the colonies. Each colony represents one CFU.
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Materials
Name | Company | Catalog Number | Comments |
5 mL polystyrene round-bottom tube | BD Falcon | 60819-138 | 12 × 75 mm style |
5% CO2 supplied incubator | Thermo Scientific | BBD6220 | |
6 mL polystyrene round-bottom tube | VWR | 89000-478 | 12 × 75 mm style with cap |
24-well plates | BD Falcon | 08-772-1H | Cell culture grade |
75 cm2 tissue culture flask | BD Falcon | BD353136 | Vented |
A549 Cell Line human | Sigma-Aldrich | 86012804-1VL | |
Cell detachment enzyme (Accutase) | Sigma-Aldrich | A6964-500ML | Cell Culture Grade |
Bovine Serum Albumins (BSA) | Sigma-Aldrich | A2058 | Suitable for cell culture |
F12 medium | Sigma-Aldrich | D6421 | Cell Culture Grade |
Fetal Calf Serum (FCS) | Sigma-Aldrich | 12003C | Suitable for cell culture |
Hemocytometer | Marienfeld | 640210 | |
Tissue culture flask | BD Falcon | 3175167 | 75 cm2 |
Vitronectin (Vn) from human plasma | Sigma-Aldrich | V8379-50UG | Cell culture grade |