Encyclopedia of Experiments: Immunology
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Transcript
Take a multi-well plate containing human precision-cut lung slices, which maintain the native lung microarchitecture.
Remove media. Add increasing test agent concentrations to the wells and incubate.
Depending on its cytotoxic potential, the test agent disrupts cell membranes, inducing cell death and reducing viable cells within the slices.
Post-incubation, remove media. Immerse the slices in media containing water-soluble tetrazolium salt, or WST-1, and an electron-coupling reagent.
Incubate. In metabolically active cells, the plasma membrane electron transport system transfers electrons from intracellular NADH to the extracellular electron-coupling reagent, reducing the cell-impermeable dye WST-1 to stable, dark-yellow, water-soluble formazan.
Post-incubation, shake the plate to evenly distribute the formazan and transfer it to a multi-well plate.
Using a microplate reader, measure the absorbance of formazan, indicative of the tissue viability.
A decrease in formazan absorbance, dependent on the test agent concentration, indicates reduced lung slice viability, confirming the cytotoxicity of the test agent.
