Encyclopedia of Experiments: Immunology
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Transcript
Begin with human precision-cut lung slices, PCLS, treated with cytotoxic agents like surfactant molecules in a multiwell plate.
The surfactant inserts into the lipid bilayer of the cell, causing the hydrophobic tails of the surfactant to interact with the hydrophobic core of the lipid bilayer.
Consequently, the orderly arrangement of the lipid molecules is disturbed, leading to the formation of a micelle.
This compromises the membrane integrity, causing the leakage of intracellular contents including, lactate dehydrogenase or LDH, a common indicator of cell membrane damage.
Collect the supernatant containing the released LDH.
Add the LDH assay reaction cocktail containing lactate substrate, NAD+, diaphorase, and tetrazolium salt, and incubate.
LDH oxidizes lactate to pyruvate, with simultaneous reduction of NAD+ coenzyme to NADH.
Diaphorase utilizes the NADH to reduce the tetrazolium salt into formazan — a colored product.
Surfactant-induced cytotoxicity can be measured from LDH activity indicated by the formazan production.
