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An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Purification of human polymorphonuclear leukocytes and isolation of human serum

NOTE: All reagents should be routinely checked for the presence of endotoxin using a commercially available endotoxin detection kit .......

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Figure 1
Figure 1: Flow cytometry analysis of purified PMNs. Representative flow cytometry dot plots of (A) purified human PMNs and (B) PMNs that have been purposely contaminated with peripheral blood mononuclear cells. (C) Representative flow cytometry histogram demonstrating minimal propidium iodide staining (<1%) of purified PMNs (shaded grey) as compared to PMNs treated with 0.05% Triton X-100 (sh.......

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