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…

Açıklamalar

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.

Etiketler

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Bu Makaleden Alıntı Yapın
Extracting Host Cell Lysate from Infected Host Cells Cultured in a Permeable Membrane System. J. Vis. Exp. (Pending Publication), e21662, doi: (2023).

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