Measuring Macrophage Phagocytic Efficiency with Primaquine and Photodynamic Therapy

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.

Begin with a suspension of a murine macrophage cell line in complete RPMI 1640 medium as mentioned in the text protocol. Dispense 100 microliters of this cell suspension into the wells of a sterile 96-well flat-bottom culture plate. Incubate the plate overnight in an incubator.

The next day, remove the plate from the incubator. Replace the spent medium with 100 microliters of fresh RPMI-1640 medium. Then, dispense 100 microliters of cryptococcal cell suspension into the culture plate containing seeded macrophages.

To study the photodynamic effect, prepare the desired working concentrations of the photosensitizer. Add 100 microliters of working solution to the plate pre-seeded with macrophages and cryptococcal cells.

Incubate the plate in the dark for the cells to react with the drug. Post-incubation, expose the plates to UV light for two minutes.

After initiating photodynamic treatment, incubate the culture plates for 18 hours. Post-incubation, remove the supernatant, and wash the cells with 200 microliters of phosphate buffer solution.

Add 300 microliters of 0.1% Triton X-100 to the wells, and incubate at room temperature for 10 minutes.

Aspirate the contents of each well and dispense them into individual sterile 1.5-milliliter plastic tubes. Dilute the cells 10 times with distilled water, and pipette 50 microliters of the dilution onto individual yeast-malt-extract agar plates.

Use a spread plate method to create a confluent lawn of cells on the surface of the agar plate. Incubate the plates for 48 hours. Count the colony-forming units on the agar plates.