Detection of Inflammasome Activation via Fluorescence Microscopy
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 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.
Protocol
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 Aseptically remove the femur and tibia from a mouse and clean the bones using sterile instruments. Place the cleaned bones in Dulbecco Modified Eagle Medium (DMEM) + 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) + 0.2 mg/mL L-glutamine + 0.05 mM 2-mercaptoethanol + 50 mg/mL …
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 | |
| 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 |
Tags
Citar este artigo
Detection of Inflammasome Activation via Fluorescence Microscopy. J. Vis. Exp. (Pending Publication), e21653, doi: (2023).