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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
- 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.
- 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.
- 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.
- 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).
- Centrifuge the 96-well plate for 5 min at 300 x g to make certain the cells are equally distributed across the well.
- Incubate the plate overnight at 37 °C + 5% CO2 before starting the priming process.
2. Priming
- 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.
- Incubate the plate for 3 h at 37 °C and 5% CO2.
3. Activation of the NLRP3 Inflammasome with Nigericin
- 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.
- 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:
- 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.
- Wash the macrophages three times for 5 min each with 1 mL of wash buffer.
- 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.
- Wash the cells three times for 5 min each with 1 mL wash buffer.
- 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.
- 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.
- 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.