Encyclopedia of Experiments: Immunology
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Transcript
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.
