Home › Analytical Chem › Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes
Analytical Chem JoVE (Open Access) Citable · DOI

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

DOI: 10.3791/60681-v
What you'll learn
  • ✓ Purify polymorphonuclear leukocytes from whole human blood samples
  • ✓ Quantify S. aureus cytotoxicity using extracellular protein assays
  • ✓ Measure bacterial cytotoxicity post-phagocytosis in immune cells
Protocol

Biopharma Insights This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

Difficulty
advanced
Total time
~4-6 hours per experiment (blood draw to final assay readout)
Model organism
Human polymorphonuclear leukocytes (primary cells)
Biosafety
BSL-2

Steps

1
Purify polymorphonuclear leukocytes from whole blood

Extract and isolate PMNs from human whole blood using density gradient centrifugation or equivalent separation methodology to obtain viable, high-purity immune cells for downstream assays.

▶ 00:44
2
Assess cytotoxicity of S. aureus extracellular proteins

Expose purified PMNs to S. aureus extracellular secreted factors and measure cell death/damage using quantitative cytotoxicity assay methodology.

▶ 04:34
3
Measure cytotoxicity following bacterial phagocytosis

Co-incubate PMNs with live S. aureus to allow phagocytosis, then quantify resulting cytotoxicity and immune cell damage using appropriate assay readouts.

▶ 06:16
4
Analyze and interpret cytotoxicity assay results

Compare quantitative cytotoxicity data between extracellular protein and phagocytosis-based assays to determine S. aureus virulence mechanisms against innate immunity.

▶ 08:40
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