This study investigates the cytotoxic effects of test agents on human precision-cut lung slices (PCLS) using a viability assay. The method involves measuring the absorbance of formazan, which indicates tissue viability post-treatment.
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.
To prepare a master mix of the working solution, dilute 25 microliters of the WST-1 reagent and 225 microliters of medium for each well. Next, pipette 250 microliters of the master-mix working solution of WST-1 for each well, and incubate the plate at 37 degrees Celsius for one hour. Ensure that the PCLS are fully covered by the WST-1 reagent during incubation.
Afterward, place the plate on an orbital shaker at 200 rpm, and shake carefully for 30 seconds to ensure thorough mixing of the WST-1 reagent. Then, pipette 100 microliters of the supernatant from each well of the 24-well plate to a new flat bottom 96-well plate in duplicates. Measure the absorption of each well at 450 nanometers using a microplate reader, and subtract the absorption at 630 nanometers reference from that at 450 nanometers.
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