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…
Divulgaciones
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.