Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes
| Autor: | Jennifer G. Dankoff, Tyler K. Nygaard, Kyler B. Pallister, Fermin E. Guerra, Alexander J. Parks, Jovanka M. Voyich, Saul Mastandrea, Kelly Gorham |
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| Rok vydání: | 2020 |
| Předmět: |
medicine.diagnostic_test
General Immunology and Microbiology Chemistry Toxin Phagocytosis General Chemical Engineering General Neuroscience chemical and pharmacologic phenomena hemic and immune systems medicine.disease_cause General Biochemistry Genetics and Molecular Biology Flow cytometry Microbiology Cell membrane chemistry.chemical_compound medicine.anatomical_structure Staphylococcus aureus medicine Centrifugation Propidium iodide Cytotoxicity circulatory and respiratory physiology |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions. |
| Databáze: | OpenAIRE |
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