Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria
In this video, we demonstrate a lysostaphin-based in vitro enzyme protection assay to quantify the Staphylococcus aureus internalization in A549 cells. The assay helps in qualitative, quantitative, and characterization studies of the intracellular bacterial load in the host cells.
Protocol
1. Cell inoculation Begin with a culture of A549 human epithelial cells in Dulbecco's modified Eagle medium (DMEM) high glucose with phenol red, supplemented with 10% fetal bovine serum (FBS) without antibiotics. Observe every well of the 24-well plate by low magnification microscopy to ensure that the human epithelial cells are healthy and growing as expected. Remove and discard the spent cell culture medium from the 24-well plate. Add 500 µL of the bacterial suspension…
Divulgazioni
The authors have nothing to disclose.
Materials
24-well plate
CORNING-FALCON
353047
A549 cell line
ATCC
CCL-185
Acetate buffer solution pH 4.6
Fluka
31048
Used to prepare lysostpahine stock solution at 10 mg/mL in 20 mM sodium acetate.
AMBICIN (Recombinant lysostaphin)
AMBI
LSPN-50
Lyophilized recominant lysostaphin. Freeze at -80 °C for long-term storage.
COS – Colombia agar + 5% sheep blood
Biomerieux
43049
Any agar plate suitable for growing staphylococci can be used instead.
Dulbecco's Modified Eagle's Medium, high glucose with phenol red
Sigma-Aldrich
D6429
Dulbecco's Modified Eagle's Medium, high glucose without phenol red
Sigma-Aldrich
D1145
Dulbecco's Phosphate Buffered Saline
Sigma-Aldrich
D8537
Dulbecco′s Phosphate-buffered Saline with MgCl2 and CaCl2, sterile-filtered
Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria. J. Vis. Exp. (Pending Publication), e21158, doi: (2023).