Overview
This video demonstrates the lactate dehydrogenase assay to detect inflammasome-mediated macrophage death. The macrophages are exposed to lipopolysaccharide for priming, followed by exposure to nigericin to cause inflammasome-induced cell death, which is determined by the lactate dehydrogenase assay.
Protocol
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.
- 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. - Incubate the plate for 3 h at 37 °C and 5% CO2.
- 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.
- 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. - 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.
- 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.
- After 30 min, add 50 μL of stop solution (1 M acetic acid) and measure OD490 using a plate reader.
- 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.
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Materials
Name | Company | Catalog Number | Comments |
Cytotox96 Non-radioactive cytotoxicity assay | Fisher Scientific | PR-G1780 | Includes Substrate Mix and Assay Buffer; Proprietary formulations. Stop solution is 1 M Acetic acid |
DMEM, high glucose, no glutamine, no phenol red | Invitrogen | 31053028 | |
Dulbecco's PBS, no calcium, no magnesium | Invitrogen | 14190144 | |
Fetal Bovine Serum, qualified, US origin | Invitrogen | 26140079 | Heat-inactivated at 55°C for 50 min |
Gentamicin | Invitrogen | 15750060 | |
Ultrapure LPS from Salmonella minnesota R595 (Re) | List Biologicals | 434 | |
Spectramax M3 plate reader | Molecular Devices | 5000414 | |
L-Glutamine | Invitrogen | 21051024 |