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

1. LDH Release Assay

NOTE: For the LDH release assay, seed the cells in a 96-well plate. Do not remove the medium after priming.

1. Replace the medium with fresh medium (DMEM-5: 10 mL of phenol-red free DMEM + 5 mM HEPES + 0.2 mg/mL L-glutamine + 0.05 mM 2-mercaptoethanol + 5% FBS) containing 100 ng/mL LPS in wells containing adherent macrophage cells (500 μL for the 24-well plates or 50 μL for the 96-well plates).
   NOTE: There are a variety of structural variations in LPS, which affect the ability to stimulate TLR4-mediated priming. LPS from Salmonella minnesota R595 (Re) is recommended.
2. Incubate the plate for 3 h at 37 °C and 5% CO₂.
3. Add 50 μL of DMEM-5 containing 10 μM nigericin to the experimental wells and 50 μL DMEM-5 to the spontaneous and 100% lysis control wells. Incubate for 60 min at 37 °C and 5% CO₂.
4. After 30 min, add 10 μL of 10x lysis buffer to the 100% lysis control wells and add 10 μL of DMEM-5 to the other wells. During this incubation, remove 1 vial of substrate mix (1.5 mL of freeze-dried substrate) and 1 aliquot of assay buffer (~12 mL) from the freezer and let them thaw protected from light to room temperature.
   NOTE: The substrate mix contains a tetrazolium salt (iodonitrotetrazolium violet), which is converted to a red formazan product.
5. After the 60 min total incubation, centrifuge the plate for 5 min at 500 x g at 10 °C. Transfer 50 μL of the supernatant to a clear flat-bottom plate. During this time, add 12 mL of assay buffer to the vial of substrate mix and incubate at room temperature for 5 min before using.
6. Add 50 μL of substrate to each well and incubate for 30 min in the dark. Include medium-only wells for blank values. Check the plate after 15 min to make sure that the signal will not exceed the detection limit of the plate reader.
7. After 30 min, add 50 μL of stop solution (1 M acetic acid) and measure OD₄₉₀ using a plate reader.
8. Calculate the percentage of cell lysis using the following formula:
   % Cytotoxicity = ((Experimental – Spontaneous)/(Maximum – Spontaneous))×100
   Experimental: nigericin-treated cells; spontaneous: untreated cells; maximum: cells exposed to lysis buffer.