Extracting Host Cell Lysate from Infected Host Cells Cultured in a Permeable Membrane System
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 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 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). Prepare overnight liquid culture…
Divulgations
The authors have nothing to disclose.
Materials
| 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. |
Tags
Citer Cet Article
Extracting Host Cell Lysate from Infected Host Cells Cultured in a Permeable Membrane System. J. Vis. Exp. (Pending Publication), e21662, doi: (2023).