Detection of Inflammasome Activation via Fluorescence Microscopy

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

1. 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 gentamicin sulfate + 10,000 U/mL penicillin/streptomycin + 10% fetal bovine serum (FBS, DMEM-10 complete) and incubate the tube on ice for 15 min.
<....

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
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