An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

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 a technique to study NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome formation in primary macrophages via immunofluorescence. The macrophages are first primed with bacterial lipopolysaccharide (LPS) to upregulate NLRP3 protein expression. Exposing the macrophages to nigericin—a potassium ionophore—leads to the activation of NLRP3 and the formation of inflammasomes, which are visualized via immunofluorescence.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Differentiation of Bone Marrow-derived Macrophages

1. Using pre-warmed differentiation medium (21 mL of DMEM-10 complete and 9 mL of L929 cell-conditioned medium as described by Swanson et al.), put bone marrow cells in a 15 cm non-tissue culture-treated Petri dish (1.2-1.5 x 10⁷ cells). Incubate the plate at 37 °C and 5% CO₂ for 7 days. Add an additional 30 mL of differentiation medium to the plate on day 3 or 4.
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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
E-MEM	ATCC	30-2003	For growing L929 cells
NCRC clone 929 (L929)	ATCC	CCL-1
Fixation/Permeabilization Kit	BD Biosciences	554714	Includes fixation and permeabilization solution, and wash buffer. Proprietary formulations. Contains 4.2% formaldehyde
Glycine	Biorad	161-0718
DMEM, high glucose, no glutamine	Invitrogen	11960044
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
ProLong Gold Mounting Medium	Invitrogen	p36934	Proprietary formulation. Curing mounting medium. Hardens to refractory index of 1.46
goat anti-mouse ALEXA555	Invitrogen	A-21422
HEPES (Ultra Pure)	Invitrogen	11344041
L-Glutamine	Invitrogen	21051024
Penicillin-Streptomycin	Invitrogen	15140122
TO-PRO-3 Iodide	Invitrogen	T3605	Far-red fluorescent nucleic acid stain
C57BL/6J mouse	Jackson Laboratoy	664
Leica SP8X Confocal Microscope	Leica
Ultrapure LPS from Salmonella minnesota R595 (Re)	List Biologicals	434
anti-ASC clone 2EI-7	Millipore-Sigma	04-417
beta-mercapto-ethanol	Millipore-Sigma	M6250-10ML
DMSO	Millipore-Sigma	D2650-5X10ML
EDTA	Millipore-Sigma	E5391