A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure
Published: October 31, 2023
Abstract
Source: den Hartigh, A.B. et al., Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages. J. Vis. Exp. (2018)
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: </str…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
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A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure. J. Vis. Exp. (Pending Publication), e21655, doi: (2023).