Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria
Published: April 30, 2023
Abstract
Source: Rigaill, J. et al. Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds. J. Vis. Exp. (2021).
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…
Divulgaciones
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 | Sigma-Aldrich | D8662 | |
| Dulbecco′s Phosphate-buffered Saline, sterile-filtered | Sigma-Aldrich | D8537 | |
| Easyspiral dilute | Interscience | 414000 | Automatic diluter and spiral plater |
| Fetal bovine serum | Gibco | 10270-106 | |
| Proteinase K, recombinant 20 mg/mL | Eurobio | GEXPRK01-B5 | > 30 U/mg, lot 901727 |
| Scan 4000 | Interscience | 438000 | Automatic colony counter |
| Sterile water | OTEC | 600500 | |
| TC20 Automated cell counter | Biorad | 1450102 | Automatic cell counter |
| Tris 1 M pH 8.0 | Invitrogen | AM9855G | Used to prepare lysostaphine working solution at 1 mg/mL in 0.1 M Tris-HCl. |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| Trypsin – EDTA solution | Sigma-Aldrich | T3924 | 0.05% porcine trypsin and 0.02% EDTA in Hanks′ Balanced Salt Solution with phenol red |
| Wide-field fluorescence microscope | Nikon | Ti2 |
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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).