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…

Divulgations

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

Tags

Play Video

Citer Cet Article
A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure. J. Vis. Exp. (Pending Publication), e21655, doi: (2023).

View Video