A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure

To perform an LDH release assay, add 50 microliters of DMEM-5 medium supplemented with 10 micromolar nigericin to each experimental well, and 50 microliters of DMEM-5 to the spontaneous, and 100% lysis control wells for 60 minutes at 37 degrees Celsius and 5% carbon dioxide. After 30 minutes add 10 microliters of 10x lysis buffer to the 100% lysis control wells, and add 10 microliters of medium to the other wells. At the end of the incubation centrifuge the plate, and transfer 50 microliters of supernatant from each well to a clear flat-bottom plate.

Next add 50 microliters of freshly thawed and prepared substrate to each well for a 30-minute incubation in the dark, checking the plate after 15 minutes to confirm that the signal will not exceed the detection limit of the plate reader. At the end of the incubation, add 50 microliters of stop solution to each well, and measure the OD₄₉₀ on an appropriate plate reader to allow calculation of the percentage of cell lysis per well.