An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

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 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 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 pl…

Declarações

The authors have nothing to disclose.

Materials

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

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An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages. J. Vis. Exp. (Pending Publication), e21654, doi: (2023).

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