The secretion system of Escherichia coli is important for the adherence and invasion of epithelial cells. I need to find out a way to measure the interaction of Escherichia coli with mammary epithelial cells in mammals. Could anyone indicate a simple, standard, and straightforward epithelial cell adhesion (and invasion) in vitro methodology? I was wondering what types of methods people usually use in this situation. Thank you very much.
A straightforward method for the evaluation of migration and invasion is the scratch wound healing assay. You can find a protocol here: Chapter Scratch Wound Healing Assay
Analysis can be done with the Image J freeware: Article An image J plugin for the high throughput image analysis of ...
You can find the software here: https://imagej.nih.gov/ij/download.html
Good luck - CL
E. coli can be added to a monolayer of epithelial cells (1 x10^5 cells/well) in a 24-well culture plate at a defined multiplicity of infection (can be variable) and incubate for 60 min (time can be variable) at 37°C in the presence of 5% CO2. Nonadherent bacteria can be removed by washing the cells after incubation. Disrupted the cells by addition of 100ul of distilled water and incubating at 37°C for 10 min. Serial dilutions of the disrupted mixture can plated on blood agar plates. E. coli colony count will help to determine the ratio of adherent cells to invading E. coli.
Alternatively, E. coli can be incubated with 0.1 mCi of [methyl-3H]thymidine for 24 h and labeled E.coli can be added to a monolayer of epithelial cells in the same fashion as mentioned above. The numbers of adhering and invading organisms can be determined using a liquid scintillation counter. The amounts of 3H detected from infected cells can be expressed as percentage of the total number of invading E. coli.