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 method to detect NLRP3 inflammasome activation in macrophages using fluorescent reporter probes. The cells are treated with potassium ionophores and glycine to induce inflammasome assembly and caspase-1 activation. Fluorescent reporter probes visualize activated caspase-1, and cells are stained with a fluorescent dye for nuclei visualization.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Harvest of Bone Marrow
2. Differentiation of Bone Marrow-derived Macrophages
3. Priming
4. Activation of the NLRP3 Inflammasome with Nigericin and Labeling with FAM-YVAD-FMK
5. Imaging of Labeled Macrophages by Confocal Microscopy
NOTE: The stained cells can be viewed after the coverslips have been allowed to cure overnight and have been sealed to the microscope slide with nail polish. Here, imaging using a confocal microscope is described. Cells can also be viewed by standard fluorescence microscopy.
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 | |
FAM-YVAD-FMK | Immunocytochemistry Technologies | 98 | |
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 |
HEPES (Ultra Pure) | Invitrogen | 11344041 | |
L-Glutamine | Invitrogen | 21051024 | |
Penicillin-Streptomycin | Invitrogen | 15140122 | |
C57BL/6J mouse | Jackson Laboratoy | 664 | |
Caspase-1/11-/- mouse | Jackson Laboratory | 16621 | |
Leica SP8X Confocal Microscope | Leica | ||
Ultrapure LPS from Salmonella minnesota R595 (Re) | List Biologicals | 434 | |
Beta-mercapto-ethanol | Millipore-Sigma | M6250-10ML | |
DMSO | Millipore-Sigma | D2650-5X10ML | |
EDTA | Millipore-Sigma | E5391 |