This study investigates the cytotoxic effects of surfactant molecules on precision-cut lung slices (PCLS). By assessing lactate dehydrogenase (LDH) release, the integrity of cell membranes can be evaluated.
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.
After incubating the PCLS with or without test agents, transfer 50 microliters of supernatant and duplicates into a new 96-well plate. This generates duplicates from each treated well of a 24-well plate.
Immediately before the assay, prepare a master-mix of the working solution of LDH reagent by thoroughly mixing 125 microliters of catalyst solution with 6.25 milliliters of dye solution for a 96-well plate. Then, pipette 50 microliters of the master-mix working solution into each well already containing 50 microliters of supernatant, and incubate the plate for 20 minutes at room temperature in the dark. Afterward, measure the absorption of each well at 492 nanometers using a microplate reader, and subtract the absorption at 630 nanometers reference from that at 492 nanometers.
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