A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins

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-based infection system to assess the impact of secreted bacterial toxins. By eliminating direct contact between the bacterial pathogen and the host cells using a permeable membrane, this technique allows for studying the effect of toxins secreted by pathogenic bacteria on mammalian host cells.

Protocol

1. Bacterial Culture Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest. Note: The GAS (Group A Streptococcus) M1T1 5448 strains utilized for these experiments included WT (Wild type) GAS', a sagAΔcat SLS-deficient (sag = Streptolysin S associated gene, SLS = Streptolysin S) mutant (abbreviated in text as ΔsagA), and a sagA complemented strain…

Materials

Todd-Hewitt broth	Acumedia	7161A	Use appropriate broth for bacterial species of interest.
BioSpectrometer	Eppendorf	basic model	Any UV-Vis spectrophotometer is suitable.
Petri Dishes	Fisher Scientific	FB0875713	Other brands are suitable.
Agar	Sigma	A1296-1kg	Other brands are suitable.
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.
10cm 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.
24-well tissue culture dishes	CytoOne	cc7682-7524	Other brands are suitable but need to be compatible with the Corning insert.
Glass coverslips	Fisherbrand	12-541-B	These must be autoclaved prior to use for cell plating.
Phosphate-Buffered Saline (PBS)	Gibco	10010-023
Phenol Red-Free DMEM	Life Technologies	31053028	Phenol Red-Free media is only required for the LDH release assay.
Bovine Serum Albumin (BSA)	Fisher Scientific	BP1600-100
L-glutamine	Gibco	25030149	Only required to supplement cell culture media for LDH release assay.
Sodium pyruvate	Gibco	BP356100	Only required to supplement cell culture media for LDH release assay.
Collagen-coated 0.4μm Transwell inserts (6 well)	Corning	3540
Collagen-coated 0.4μm Transwell inserts (24 well)	Corning	3595
Forceps	Fisher	3120018	These must be sterilized with ethanol prior to handeling Transwell inserts.
Cell scrapers	Fisherbrand	08-100-241	These are only required for the collection of cell lysates.