This study investigates the interaction between macrophages and Cryptococcus neoformans, focusing on the phagocytosis process and subsequent cellular effects. The research highlights how fungi can survive and replicate within macrophages, leading to cellular damage and lysis.
Take a multi-well plate containing macrophages.
Add Cryptococcus neoformans to the test wells, while the control wells contain only macrophages.
Macrophages phagocytose fungi within phagosomes.
Wash with buffer to remove non-phagocytosed fungi. Add antibiotic-free media. Incubate.
The phagosome merges with the lysosome, forming a phagolysosome. Fungi modulate phagolysosome pH and possess intrinsic mechanisms to survive harsh phagolysosomal conditions. Fungi permeabilize the phagolysosomal membrane and replicate, accompanied by cytosolic accumulation of polysaccharide-filled vesicles.
Intracellular fungal residence and replication cause cellular damage, triggering cell-death pathways. This causes macrophage lysis and intracellular content release, including lactate dehydrogenase or LDH, into media.
Collect LDH-containing media at different time points. Lyse the control macrophages at similar time points for maximum cellular LDH release. Add the assay mixture to the collected media and lysates. LDH converts lactate to pyruvate, reducing NAD+. Diaphorase reduces tetrazolium salt, forming a red formazan product.
Using a plate reader, measure the formazan absorbance to quantify macrophage lysis following Cryptococcus neoformans infection.
To prepare fungal cells for a macrophage infection, collect and centrifuge C. neoformans cells from a mid-log phase culture, followed by three gentle washes with 1 milliliter of room temperature PBS under the same centrifuge conditions.
After the last wash, re-suspend the fungal cells at a 1.2 times 10 to the eighth cells per milliliter, of antibiotic-free cell culture medium concentration, and add 1 milliliter of fungal cell suspension to each of 4 wells of a 70% to 80% confluent 6-well macrophage culture plate. Then, place the plate in the cell culture incubator for three hours. Carefully tilt the plate to allow each well to be gently washed three times with 1 milliliter of PBS per well per wash.
To measure the infection proficiency after the last wash, add 1 milliliter of antibiotic-free medium to each well of the 6-well plate, and place the plate in the cell culture incubator. At the appropriate experimental time points, collect the supernatant from each well, and measure the amount of LDH in each well according to standard protocols. At the same time points, lyse the uninfected macrophage cells to determine the value for maximum cytotoxicity.
The cytotoxicity can then be calculated using the formula as indicated.
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