Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy
Transcrição
Begin with a multi-well plate containing adhered murine macrophages — or phagocytic immune cells.
Introduce a fungal cell suspension of Cryptococcus, interacting with macrophage receptors and internalizing via phagocytosis.
The phagosome fuses with the lysosome, forming a phagolysosome. The acidic environment of the phagolysosome favors fungal survival and multiplication.
Introduce a high concentration of primaquine — a photosensitizer, to the test wells, while the control lacks the photosensitizer. Incubate to facilitate primaquine uptake by macrophages.
Expose the plate to ultraviolet light for photodynamic treatment or PDT.
In the test well, primaquine forms radical cations that interact with molecular oxygen, producing reactive oxygen species, or ROS.
ROS induce fungal death, while anti-oxidative enzymes of macrophages neutralize ROS, preventing cellular damage.
Permeabilize the macrophages, releasing fungal cells.
Spread the diluted cell contents on selective agar plates, yielding distinct fungal colonies.
Fewer colonies on the test plate compared to the control indicate the enhanced macrophage phagocytic efficiency due to photodynamic treatment with primaquine.
