Encyclopedia of Experiments: Immunology
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Transcript
Take a multi-well plate containing adherent macrophages.
Add serially diluted test photosensitizer to test wells. The control well lacks the photosensitizer.
Incubate for cellular uptake of the photosensitizer.
Expose the plate to UV light, termed photodynamic treatment, or PDT, for a specified duration.
In the test wells, photosensitizers within the macrophages absorb UV light and become excited.
These excited photosensitizers react with molecular oxygen, forming reactive oxygen species, or ROS, and causing cellular toxicity.
To counteract this, cellular antioxidant enzymes, including superoxide dismutase and catalase, neutralize ROS, preventing cellular oxidative damage.
Add XTT reagent, a tetrazolium salt, and an electron-coupling reagent to both test and control wells. Incubate.
In metabolically active cells, the trans-plasma membrane electron transport system transfers electrons from mitochondrial NADH to the electron-coupling reagent, reducing XTT to an orange formazan product.
Comparable formazan absorbance in test and control wells indicates that PDT exposure with photosensitizer treatment does not impact macrophage metabolic activity.
