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…

Divulgaciones

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

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An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells. J. Vis. Exp. (Pending Publication), e21696, doi: (2023).

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