Extracting Host Cell Lysate from Infected Host Cells Cultured in a Permeable Membrane System

Abstract

Source: Flaherty, R. A., et al. Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells. J. Vis. Exp. (2016).

This video demonstrates a permeable membrane insert system to investigate the impact of bacterial toxins on host cells in a controlled and isolated manner. This approach allows for the selective exposure of the cells to the toxins while maintaining a physical separation that mimics the natural host-pathogen interaction.

Protocol

1. Bacterial Culture

1. Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest.
   Note: The GASM1T1 5448 Streptococcus strains utilized for these experiments included WT GAS', a sagAΔcat SLS-deficient mutant (abbreviated in the text as ΔsagA), and a sagA complemented strain (abbreviated in the text as ΔsagA+sagA).
2. Prepare overnight liquid cultures of bacterial strains of interest.  Grow GAS M1T1 5448 strains in  -10 ml of Todd-Hewitt broth at 37°C for 16-20 hr prior to infecting hum....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HaCaT human epithelial keratinocytes			Gift from lab of Victor Nizet.
Dulbecco's Modified Eagle's Medium (DMEM)	Life Technologies	11995-073	Use appropriate media for cell line of interest.
Fetal Bovine Serum (FBS)	Biowest	S162H	Use appropriate media additives for cell line of interest.
10 cm tissue culture dishes	Nunc	150350	Other brands are suitable.
6-well tissue culture dishes	CytoOne	cc7682-7506	Other brands are suitable but need to be compatible with the Corning insert.
Collagen-coated 0.4 μm Transwell inserts (6 well)	Corning	3540
Collagen-coated 0.4 μm Transwell inserts (24 well)	Corning	3595
Cell scrapers	Fisherbrand	08-100-241	These are only required for the collection of cell lysates.
Nonidet P40 Substitute	Sigma	74385-1L	Other suppliers are suitable.