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

Published: October 31, 2023

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…

Açıklamalar

The authors have nothing to disclose.

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.

Etiketler

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Bu Makaleden Alıntı Yapın
A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins. J. Vis. Exp. (Pending Publication), e21661, doi: (2023).

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