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

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.

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 minnesot....

Materials

List of materials used in this article

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