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…
Offenlegungen
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. |
Tags
Diesen Artikel zitieren
A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins. J. Vis. Exp. (Pending Publication), e21661, doi: (2023).