Encyclopedia of Experiments: Immunology
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Transcript
Begin with tubes containing human whole blood, including red blood cells, or RBCs, and white blood cells, or WBCs, such as monocytes and granulocytes, and other blood cells.
Introduce dihydroethidium — a superoxide indicator and unlabeled bacteria into one tube. Treat the other tube with green fluorophore-labeled bacteria.
Granulocytes and monocytes interact with the bacteria's pathogen-associated molecules, engulfing them within phagosomes.
This activates the phagosomal membrane enzyme NADPH oxidase, reducing molecular oxygen to a superoxide anion.
The superoxide anion further produces hydrogen peroxide — a reactive-oxygen species, or ROS resulting in an oxidative burst.
Within the phagosome, ROS oxidizes dihydroethidium to form 2-hydroxy ethidium, which stains bacterial DNA, imparting a red fluorescence.
Introduce a quencher, to quench the extracellular fluorescence.
Add a lysing solution, eliminating potential RBC interference, and fix the WBCs.
In flow cytometry, cells with green fluorescence confirm granulocytes' and monocytes' phagocytic activity, while cells with red fluorescence in another tube indicate oxygen-burst activity in these cells.
