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.
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
2. Priming
3. Activation of the NLRP3 Inflammasome with Nigericin
4. Antibody Staining
NOTE: To label the cells with antibodies to detect ASC, seed, prime and expose the cells to nigericin identical to the previous section. The processing afterward is as follows:
The authors have nothing to disclose.
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 |