This study investigates the effect of drug treatment on the phagocytic ability of macrophages against Cryptococcus neoformans. By utilizing a pH-sensitive fluorescent dye, the research demonstrates how drug molecules enhance macrophage function in internalizing fungal cells.
Begin with a fungal suspension of Cryptococcus. Add a solution of fungal-derived bioparticles conjugated with a pH-sensitive fluorescent dye to stain the Cryptococcus cells.
Wash to remove the unbound dye. Transfer the stained fungal suspension into a multi-well plate with adhered macrophages — or phagocytic immune cells.
Introduce the minimum inhibitory concentration of a drug, such as aspirin or ibuprofen, and incubate to facilitate the drug uptake.
The drug molecules interact with cyclooxygenase-2 — a prostaglandin-synthesizing enzyme, inhibiting its activity.
This prevents the conversion of arachidonic acid to prostaglandin, preserving intracellular cAMP levels and enhancing the macrophage phagocytic ability, facilitating the internalization of fungi.
The fungi-containing phagosome fuses with the lysosome — an acidic organelle, forming a phagolysosome with an acidic pH and allowing the dye to fluoresce.
Drug-treated cells exhibit a higher fluorescence than untreated cells, suggesting the drug’s efficacy in enhancing the phagocytic ability of macrophages.
Begin with a culture of the murine macrophage cell line, grown in complete RPMI 1640 medium, as described in the text protocol.
Additionally, prepare a culture of Cryptococcus neoformans resuspended in PBS to the desired concentration, as mentioned in the text protocol.
To stain the cryptococcal cells with the phagocytosis stain, dispense 999 microliters of the cells into a microcentrifuge tube. Add 1 microliter of the stain to the tube. Incubate the cells for 1 hour at room temperature, in the dark, with slow agitation.
After incubation, wash the cells by first centrifugation. Then, discard the supernatant. Resuspend the cells with PBS and repeat the washing step two additional times.
Dispense 100 microliters of the Cryptococcus neoformans cell suspension into the wells of the 96-well plate containing the cultured macrophages.
To study the effect of the test drugs on macrophage phagocytosis, dispense 100 microliters of the desired test drug into appropriate wells of the 96-well plate as described in the text protocol.
Incubate the plate in a carbon dioxide incubator for 2 to 6 hours. At the end of the incubation, measure the fluorescence from the phagocytic stain.
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