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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