An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells
Published: September 29, 2023
Abstract
Source: Singh, B. et al., Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance. J. Vis. Exp. (2018)
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 mic…
Divulgations
The authors have nothing to disclose.
Materials
| 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 |
Tags
Citer Cet Article
An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells. J. Vis. Exp. (Pending Publication), e21696, doi: (2023).