All microscopy was done at the W. M. Keck Microscopy Center with support of Nathaniel Peters and with support of NIH award S10OD016240. S.L.F. is supported by NIH K08AI119142 and R21AI130281. We thank Dr. Richard Flavell and Dr. Brad Cookson for caspase-1/11 deficient mice, and Dr. Brad Cookson for sharing laboratory facilities and equipment.

All animal procedures were approved and conducted under University of Washington Institutional Animal Care and Use Committee guidelines.
1. Harvest of Bone Marrow
<ol>
	<li>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 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 (...

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	<li>Jorgensen, I., Miao, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyroptotic+cell+death+defends+against+intracellular+pathogens.">Pyroptotic cell death defends against intracellular pathogens.</a> Immunol Rev. 265 (1), 130-142 (2015).</li><li>Guo, H., Callaway, J. B., Ting, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammasomes:+mechanism+of+action,+role+in+disease,+and+therapeutics.">Inflammasomes: mechanism of action, role in disease, and therapeutics.</a> Nat Med. 21 (7), 677-687 (2015).</li><li>Bergsbaken, T., Fink, S. L., Cookson, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyroptosis:+host+cell+death+and+inflammation.">Pyroptosis: host cell death and inflammation.</a> Nat Rev Microbiol. 7 (2), 99-109 (2009).</li><li>Martinon, F., Burns, K., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+molecular+platform+triggering+activation+of+inflammatory+caspases+and+processing+of+proIL-beta.">The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.</a> Mol Cell. 10 (2), 417-426 (2002).</li><li>Fink, S. L., Cookson, B. 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S., Vakifahmetoglu-Norberg, H., Yuan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deubiquitination+of+NLRP3+by+BRCC3+critically+regulates+inflammasome+activity.">Deubiquitination of NLRP3 by BRCC3 critically regulates inflammasome activity.</a> Mol Cell. 49 (2), 331-338 (2013).</li><li>Song, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NLRP3+Phosphorylation+Is+an+Essential+Priming+Event+for+Inflammasome+Activation.">NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation.</a> Mol Cell. 68 (1), 185-197 (2017).</li><li>Stutz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NLRP3+inflammasome+assembly+is+regulated+by+phosphorylation+of+the+pyrin+domain.">NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain.</a> J Exp Med. 214 (6), 1725-1736 (2017).</li><li>Broz, P., Dixit, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammasomes:+mechanism+of+assembly,+regulation+and+signalling.">Inflammasomes: mechanism of assembly, regulation and signalling.</a> Nat Rev Immunol. 16 (7), 407-420 (2016).</li><li>Masumoto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASC,+a+novel+22-kDa+protein,+aggregates+during+apoptosis+of+human+promyelocytic+leukemia+HL-60+cells.">ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells.</a> J Biol Chem. 274 (48), 33835-33838 (1999).</li><li>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASC+is+essential+for+LPS-induced+activation+of+procaspase-1+independently+of+TLR-associated+signal+adaptor+molecules.">ASC is essential for LPS-induced activation of procaspase-1 independently of TLR-associated signal adaptor molecules.</a> Genes Cells. 9 (11), 1055-1067 (2004).</li><li>Miao, E. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+cell+lysis+are+separable+events+during+pyroptosis.">Cell death and cell lysis are separable events during pyroptosis.</a> Cell Death Discov. , (2017).</li><li>Russo, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+Caspase-1+Induces+Plasma+Membrane+Pores+That+Precede+Pyroptotic+Lysis+and+Are+Blocked+by+Lanthanides.">Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides.</a> J Immunol. 197 (4), 1353-1367 (2016).</li><li>Evavold, C. 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In this manuscript, we have presented two techniques to examine inflammasome activation and the consequence of caspase-1 activation following NLRP3 stimulation in murine bone marrow-derived macrophages. The first technique allows the researcher to determine the level of caspase-1 activation on a cellular basis using the fluorescent reporter FAM-YVAD-FMK. This reporter is highly specific for binding the caspase-1 family of enzymes, as seen by the absence of staining in caspase-1/11 deficient macrophages. Specificity was a...

Inflammasome-mediated inflammation is a critical component of the defense against pathogenic organisms1, but also underlies the etiology of many diseases2. The inflammatory response to a wide range of infections is triggered by cytosolic detection of pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs). Pattern recognition receptors (PRR), including members of the NOD-like receptor (NLR) family, oligomerize upon the detection of these PAMPs and DAMPs. This triggers the formation of a multi-protein complex termed the inflammasome, which contains the PRR, the adaptor protein Apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain (CARD) (ASC), and the pro-form of caspase-13,4. This complex allows proximity-induced auto-activation of caspase-1. Active caspase-1 then leads to a set of events that are characteristic of the inflammatory cell death pathway pyroptosis. These events include: the cleavage and release of the inflammatory cytokines IL-1&#946; and IL-18, lysosome exocytosis with release of lysosomal contents into the extracellular space, nuclear condensation, and gasdermin D cleavage. The released N-terminal domain of gasdermin D inserts into the plasma membrane, forming pores that cause plasma membrane rupture and release of inflammatory contents, in addition to taking away a protective niche for pathogen replication5,6,7.
Here, we focus on the well-studied NLRP3 inflammasome. The activation of the NLRP3 inflammasome occurs through a two-step process8. The first &#34;priming&#34; step occurs through the recognition by Toll-like receptors (TLR) of a microbial product. This is replicated in the laboratory setting by using LPS to stimulate TLR4. This stimulation upregulates NLRP3 and pro-IL-1&#946; through NF-kB signaling. Priming additionally licenses NLRP3 through non-transcriptional mechanisms by inducing its deubiquitination9,10 and phosphorylation or dephosphorylation of specific residues11,12.
The second signal for NLRP3 activation is thought to involve mitochondrial factors, reactive oxygen species, potassium efflux and calcium signaling, although a unifying mechanism for NLRP3 activation remains elusive8. Activated NLRP3 oligomerizes through interactions between its NACHT domains, and recruits ASC via binding of the pyrin (PYD) domains13. In each cell, these macromolecular complexes form a single microscopically visible focus. ASC was originally identified as a 22 KDa protein that forms a &#34;speck&#34; during the apoptosis of human leukemia cells, and named apoptosis-associated speck-like protein containing a CARD14. It was later determined that ASC recruits pro-caspase-1 through the interaction of the CARD of ASC with the CARD of pro-caspase-1, forming the inflammasome15.
Not all NLRs require the presence of ASC to induce caspase-1 activation. Unlike NLRP3, NLRC4 and NLRP1b have CARD domains and can directly recruit pro-caspase-1 via CARD-CARD interactions to induce caspase-1 activation. In the absence of ASC, active caspase-1 remains diffuse throughout the cytosol and does not form a single focus. This diffuse active caspase-1 is sufficient to induce pyroptotic cell death, but is unable to process pro-IL-1&#946;13,16.
In this manuscript, we will discuss two ways to assess inflammasome activation. The first uses the fluorescent activity probe, FAM-YVAD-FMK, which binds the caspase-1 family of proteases. This family includes caspase-1, but also mouse caspase-11 and human caspase-4 and caspase-5. By using macrophages from caspase-1/11 deficient mice in all experiments, the specificity of this probe will be addressed. This method can be combined with antibody labeling of inflammasome components, which we will also describe. Microscopic visualization allows for the identification of individual cells containing active caspase-1 and oligomerized ASC. Using this method, researchers will be able to determine where in the inflammasome formation cascade their manipulations of the host cell or the pathogenic organism under study have an effect. For example, one can distinguish whether a given intervention prevents the recruitment of ASC to the NLRP3 complex or targets the subsequent recruitment and activation of caspase-117. Examining outcomes of caspase-1 activation such as IL-1&#946; secretion would not be able to distinguish between these two possibilities. Also, the secretion of IL-1&#946; could be altered without altering the cells ability to activate caspase-1 and undergo pyroptotic cell death16.
The second method measures the release of lactate dehydrogenase (LDH) into the cell supernatant, which occurs during pyroptotic macrophage lysis following caspase-1 activation as described above. This second method is a population-based approach as the release of LDH in the entire well is measured. This simple approach allows for the rapid analysis (30 min of incubation time) of samples to determine if there is caspase-1-mediated cell death and can be performed in a 96-well plate format.
These methods are complementary, each with different advantages, and both are easily amenable to modifications. For example, macrophage treatment with targeted small molecular weight compounds prior to inflammasome stimulation can be used to investigate the role certain proteins may have on controlling inflammatory responses.

<table>
	<tbody>
		<tr>
			<td>E-MEM</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>For growing L929 cells</td>
		</tr>
		<tr>
			<td>NCRC clone 929 (L929)&#160;</td>
			<td>ATCC</td>
			<td>CCL-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Kit</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Includes fixation and permeabilization solution, and wash buffer.&#160; Proprietary formulations. Contains 4.2% formaldehyde</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotox96 Nonradioactive cytotoxicity assay</td>
			<td>Fisher Scientific</td>
			<td>PR-G1780</td>
			<td>Includes Substrate Mix and Assay Buffer; Proprietary formulations. Stop solution is 1 M Acetic acid</td>
		</tr>
		<tr>
			<td>FAM-YVAD-FMK</td>
			<td>Immunocytochemistry Technologies</td>
			<td>98</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, no glutamine</td>
			<td>Invitrogen</td>
			<td>11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, no glutamine, no phenol red</td>
			<td>Invitrogen</td>
			<td>31053028</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS, no calcium, no magnesium</td>
			<td>Invitrogen</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, US origin</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>Heat inactivated at 55&#176;C for 50 min</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Mounting Medium</td>
			<td>Invitrogen</td>
			<td>p36934</td>
			<td>Proprietary formulation. Curing mounting medium. Hardens to refractory index of 1.46</td>
		</tr>
		<tr>
			<td>goat anti-mouse ALEXA555</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (Ultra Pure)</td>
			<td>Invitrogen</td>
			<td>11344041</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>21051024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-PRO-3 Iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>far-red fluorescent nucleic acid stain&#160;</td>
		</tr>
		<tr>
			<td>C57BL/6J mouse</td>
			<td>Jackson Laboratoy</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>caspase-1/11-/- mouse</td>
			<td>Jackson Laboratory</td>
			<td>016621</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8X Confocal Microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure LPS from Salmonella minnesota R595 (Re)</td>
			<td>List Biologicals</td>
			<td>434</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ASC clone 2EI-7</td>
			<td>Millipore-Sigma</td>
			<td>04-417</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-mercapto-ethanol</td>
			<td>Millipore-Sigma</td>
			<td>M6250-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Millipore-Sigma</td>
			<td>D2650-5X10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Millipore-Sigma</td>
			<td>E5391</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectramax M3 plate reader</td>
			<td>Molecular Devices</td>
			<td>5000414</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of inflammasome activation and pyroptotic cell death in murine bone marrow-derived macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

To detect caspase-1 activation following the exposure to nigericin, the cells were processed as described in the protocol section. We combined both FAM-YVAD-FMK and ASC antibody labeling in order to show that ASC and active caspase-1 co-localize following NLRP3-mediated inflammasome activation. Figure 1A shows that wild type macrophages exposed to 5 &#956;M nigericin have formation of an ASC focus in the perinuclear region. These foci also contain active casp...

Watch this Scientific Journal Video about Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages at JoVE.com

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

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.

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Research

JoVE Journal

Immunology and Infection

10.4K Views.  University of Washington. 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.Demonstrat...