An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells

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

1. 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.
   1. Wash the cell monolayer in the flask twice ....

Materials

List of materials used in this article

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