The overall goals of this protocol are to examine inflammasome activation at the single cell level via fluorescence microscopy and to measure lysis during pyroptosis by a lactate dehydrogenase, or LDH release assay. This method can help answer key questions in the inflammasome field, such as how is Caspase-1 activation regulated. The main advantage of these techniques is that they allow the user to examine multiple steps of the inflammasome activation and pyroptosis pathways.
Demonstrating the procedure will be Andreas den Hartigh, a research scientist from my laboratory. Begin by replacing the medium from LPS-primed bone marrow derived macrophages seeded on glass coverslips with 290 microliters of DMEM 5 medium, supplemented with five micromolar nigericin and five millimolar glycine, per well. Return the 24-well plate to the cell culture incubator for 60 minutes at 37 degrees Celsius and 5%carbon dioxide, adding 10 microliters of 30X FAM-YVAD-FMK after the first 15 minutes.
At the end of the incubation, wash the cells with one milliliter of cold PBS per well, three times for five minutes per wash and fix the cells with 250 microliters of 2%paraformaldehyde per well for 30 minutes on ice protected from light, labeling the cells with an appropriate fluorescence nuclear dye during the last five minutes. At the end of the incubation, wash the cells three times with cold PBS as just demonstrated and load each slide with seven microliters of anti-fade mounting medium. Place a coverslip onto each slide and allow the mounting medium to harden overnight before sealing the coverslips with nail polish.
To image the cells by fluorescence confocal microscopy, place the untreated control slide onto the microscope stage and manually focus on the cells. Using the microscope look up table settings, adjust the offset until no positive probe staining is observed in the untreated cells. Next, using a nigericin treated sample, locate a field that contains cells that are both positive and negative for the probe staining and adjust the imaging plane of the probe channel to the plane that has the highest intensity of positive staining.
Then adjust the gain until the foci are visible in the cells with condensed nuclei and obtain images of five randomly selected fields per slide at a 100X total magnification. For immunofluorochemical analysis of the nigericin treated cells, wash the prop-labeled cells as just demonstrated and incubate the cells in 250 microliters of fixation and permeabilization solution per well for 30 minutes on ice, protected from light. Wash the macrophages three more times with fresh wash buffer and label the cells with 250 microliters of primary antibody against a ptosis associated speck-like protein containing a C-terminal caspase-recruitment domain, or ASC, per well, for one hour on ice.
Wash the cells at the end of the incubation and label the samples with 250 microliters of the appropriate fluorescence dye conjugated secondary antibody for another hour on ice, adding four microliters of an appropriate fluorescence nuclear staining dye during the last five minutes. After washing the cells three times in cold wash buffer, mount coverslips onto the slides with seven microliters of anti-fade mounting medium, as demonstrated, and image the macrophages by fluorescence confocal microscopy. To perform an LDH release assay, add 50 microliters of DMEM 5 medium supplemented with 10 micromolar nigericin to each experimental well and 50 microliters of DMEM 5 to the spontaneous and 100%lysis control wells for 60 minutes at 37 degrees Celsius and 5%carbon dioxide.
After 30 minutes, add 10 microliters of 10X lysis buffer to the 100%lysis control wells and add 10 microliters of medium to the other wells. At the end of the incubation, centrifuge the plate and transfer 50 microliters of supernatant from each well to clear, flat-bottomed plate. Next, add 50 microliters of freshly thawed and prepared substrate to each well for a 30 minute incubation in the dark, checking the plate after 15 minutes to confirm that the signal will not exceed the detection limit of the plate reader.
At the end of the incubation, add 50 microliters of stop solution to each well and measure the OD490 on an appropriate plate reader to allow calculation of the percentage of cell lysis per well. While type macrophages exposed to nigericin demonstrate the formation of an ASC focus in the perinuclear region that also contain active Caspase-1, indicating that these cells are undergoing pyroptosis. While macrophages from Caspase-111 deficient mice also generate an ASC focus, these cells do not have any active Caspase-1 associated with this focus, as indicated by the absence of FAM-YVAD-FMK staining.
Nor are the nuclei of these cells condensed, indicating an absence of pyroptotic cell death. Further, a large percentage of wild type macrophages undergo cell death after nigericin exposure, as measured by LDH release analysis of their culture supernatants, while macrophages deficient in Caspase-1 do not release LDH into the supernatant, indicating that the cells are still intact. After watching this video, you should have a good understanding of how to visualize inflammasome formation by fluorescence microscopy and to determine the level of cell lysis by measuring LDH release.
This procedure can be modified for the use of other inflammasome stimuli or interventions that alter inflammasome activation or pyroptosis.
