An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

Protocolo

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

1. 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 plate at 37 °C and 5% CO₂ for 7 days. Add an additional 30 mL of differentiation medium to the plate on day 3 or 4.
   NOTE: It is important not to use tissue culture-treated plates, as macrophages will be difficult to detach and harvest.
2. To collect macrophages, wash the plate with phosphate-buffered saline (PBS) and add 20 mL of cold PBS + 1 mM ethylenediaminetetraacetic acid (EDTA). Incubate the plate at 4 °C for 10 min. Harvest the cells by pipetting the PBS + EDTA over the cells and washing the plate with 10 mL of PBS. Combine both washes into two 15 mL conical tubes.
3. Centrifuge the cells at 300 x g for 10 min and resuspend the cells in 10 mL of phenol-red free DMEM + 5 mM HEPES + 0.2 mg/mL L-glutamine + 0.05 mM 2-mercaptoethanol + 5% FBS (DMEM-5) and count the cells using either a hemocytometer or a Coulter counter. Keep the cells on ice during the counting.
4. Seed the macrophages in tissue culture-coated plates at 2 x 10⁵ cells/mL. For microscopy experiments, seed 1 mL of cells on coverslips in 24 well plates. For LDH release assays, seed 100 µL of cells in a 96-well plate. For the LDH assay, seed 9 wells (3 wells untreated, 3 wells with 100% lysis controls, and 3 wells for the experimental stimulus).
5. Centrifuge the 96-well plate for 5 min at 300 x g to make certain the cells are equally distributed across the well.
6. Incubate the plate overnight at 37 °C + 5% CO₂ before starting the priming process.

2. Priming

1. Replace the medium with fresh medium (DMEM-5) containing 100 ng/mL LPS (500 μL for the 24-well plate or 50 μL for the 96-well plate).
   NOTE: There are a variety of structural variations in LPS, which affect the ability to stimulate TLR4-mediated priming. LPS from Salmonella minnesota R595 (Re) is available from multiple vendors and is recommended.
2. Incubate the plate for 3 h at 37 °C and 5% CO₂.

3. Activation of the NLRP3 Inflammasome with Nigericin

1. Remove the medium and replace it with 290 μL of DMEM-5 containing 5 μM nigericin and 5 mM glycine. Glycine is added to reduce the amount of cell lysis that occurs during caspase-1 activation. Incubate for 60 min at 37 °C and 5% CO₂. Use both wild-type and caspase-1 deficient macrophages to ensure that the observed labeling is specific for caspase-1.
2. After 60 min, remove the medium and wash the cells three times for 5 min each with 1 mL of cold PBS.

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:

1. Wash the cells three times for 5 min each with 1 mL of cold PBS. Add 250 μL of fixation and permeabilization solution. Incubate the cells on ice, and cover for 30 min.
2. Wash the macrophages three times for 5 min each with 1 mL of wash buffer.
3. Add 250 μL of primary antibody against ASC diluted 1:500 in wash buffer and incubate for 1 h on ice. Include one coverslip that does not receive any primary antibody, but will only receive the secondary. This coverslip will be used during the microscope setup to set the correct offset.
4. Wash the cells three times for 5 min each with 1 mL wash buffer.
5. Add 250 μL of secondary antibody (fluorescent dye conjugated goat-anti-mouse) diluted 1:500 in wash buffer and incubate for 1 h on ice. Add 4 μL of 0.2 mM far-red fluorescent nucleic acid stain for the last 5 min of this incubation to label nuclei.
6. Wash the cells 3x with 1 mL of cold wash buffer and mount the coverslips onto microscope slides using a 7 μL anti-fade mounting medium. Let the mounting medium harden overnight before sealing the coverslip to the microscope slide using clear nail polish.
7. Image macrophages by confocal microscopy. Ex/Em for the secondary antibody is 555/580 nm and 642/661 for the far-red fluorescent nucleic acid stain.

Materiais

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
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