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 (FBS, DMEM-10 complete) and incubate the tube on ice for 15 min.</li>
	<li>Remove the proximal and distal ball joints using scissors. Insert a 25 G needle attached to a 10 mL syringe filled with medium into the hollow of the bones. Flush out the bone marrow using DMEM-10 complete. Resuspend any clumps by pipetting the medium up and down gently.</li>
	<li>Centrifuge the cells for 10 min at 300 x g and count the number of cells using a hemocytometer. At this point, either freeze the cells using 90% FBS + 10% dimethyl sulfoxide (DMSO) or plate the cells in a 15 cm Petri dish to differentiate macrophages using L929 conditioned medium (step 2.1).</li>
</ol>
2. Differentiation of Bone Marrow-derived Macrophages
<ol>
	<li>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.18), 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 &#176;C and 5% CO2 for 7 days. Add an additional 30 mL of differentiation medium to the plate at day 3 or 4. 
	NOTE: It is important not to use tissue culture treated plates, as macrophages will be difficult to detach and harvest.</li>
	<li>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 &#176;C for 10 min. Harvest the cells by pipetting the PBS + EDTA over the cells and wash the plate with 10 mL of PBS. Combine both washes into two 15 mL conical tubes.</li>
	<li>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.</li>
	<li>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 &#181;L 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).</li>
	<li>Centrifuge the 96 well plate for 5 min at 300 x g to make certain the cells are equally distributed across the well.</li>
	<li>Incubate the plate overnight at 37 &#176;C + 5% CO2 before starting the priming process.</li>
</ol>
3. Priming
<ol>
	<li>Replace the medium with fresh medium (DMEM-5) containing 100 ng/mL LPS (500 &#956;L for the 24 well plate or 50 &#956;L for the 96 well plate). 
	NOTE: There are a variety of structural variations in LPS, which affect the ability to stimulate TLR4-mediated priming19. LPS from Salmonella minnesota R595 (Re) is available from multiple vendors and is recommended.</li>
	<li>Incubate the plate for 3 h at 37 &#176;C and 5% CO2.</li>
</ol>
4. Activation of the NLRP3 Inflammasome with Nigericin and Labeling with FAM-YVAD-FMK
<ol>
	<li>Remove the medium and replace it with 290 &#956;L of DMEM-5 containing 5 &#956;M nigericin and 5 mM glycine. Glycine is added to reduce the amount of cell lysis that occurs during caspase-1 activation5. Incubate for 60 min at 37 &#176;C and 5% CO2. Use both wild type and caspase-1 deficient macrophages to ensure that the observed labeling is specific for caspase-1.</li>
	<li>During the last 45 min, add 10 &#956;L of 30x FAM-YVAD-FMK, prepared according to the manufacturer&#39;s instructions.</li>
	<li>After 60 min, remove the medium and wash the cells three times for 5 min each with 1 mL of cold PBS.</li>
	<li>Add 250 &#956;L of 2% paraformaldehyde to each well and incubate on ice covered with aluminum foil for 30 min.</li>
	<li>During the last 5 min, add 4 &#956;L of 0.2 mM far-red fluorescent nucleic acid stain to label the nuclei. 4&#39;,6-diamidino-2-phenylindole (DAPI) or other dyes can also be used to label DNA.</li>
	<li>Wash the cells three times with 1 mL of cold PBS and mount the coverslips onto microscope slides using 7 &#956;L anti-fade mounting medium. Let the mounting medium harden over night before sealing the coverslip to the microscope slide using nail polish.</li>
	<li>Image the macrophages by confocal microscopy. Ex/Em for FAM-YVAD-FMK is 492/520nm and 642/661 for far-red fluorescent nucleic acid stain. Make sure to set up the microscope such that untreated cells do not show any background staining in the 488 channel. Otherwise, background FAM-YVAD-FMK staining will be detected and not actual active caspase-1. 
	NOTE: Microscope set-up is described in section 6.</li>
</ol>
5. 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 afterwards is as follows:
<ol>
	<li>Wash the cells three times for 5 min each with 1 mL of cold PBS. Add 250 &#956;L of fixation and permeabilization solution. Incubate the cells on ice, and covered for 30 min.</li>
	<li>Wash the macrophages three times for 5 min each with 1 mL of wash buffer.</li>
	<li>Add 250 &#956;L 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.</li>
	<li>Wash the cells three times for 5 min each with 1 mL wash buffer.</li>
	<li>Add 250 &#956;L of secondary antibody (fluorescent dye conjugated goat-anti-mouse) diluted 1:500 in wash buffer and incubate for 1 h on ice. Add 4 &#956;L of 0.2 mM far-red fluorescent nucleic acid stain for the last 5 min of this incubation to label nuclei.</li>
	<li>Wash the cells 3x with 1 mL of cold wash buffer and mount the coverslips onto microscope slides using 7 &#956;L anti-fade mounting medium. Let the mounting medium harden over night before sealing the coverslip to the microscope slide using clear nail polish.</li>
	<li>Image macrophages by confocal microscopy. Ex/Em for the secondary antibody is 555/580 nm and 642/661 for far-red fluorescent nucleic acid stain (as described below). 
	NOTE: Labeling with FAM-YVAD-FMK and antibody labeling can be combined to show co-localization of active caspase-1 and ASC. For this, follow the conditions for FAM-YVAD-FMK labeling and then switch to the antibody labeling protocol for further processing.</li>
</ol>
6. Imaging of Labeled Macrophages by Confocal Microscopy
NOTE: The stained cells can be viewed after the coverslips have been allowed to cure overnight and have been sealed to the microscope slide with nail polish. Here, imaging using a confocal microscope is described. Cells can also be viewed by standard fluorescence microscopy.
<ol>
	<li>Place the slide with the untreated control cells on the microscope stage and focus the microscope on the cells.</li>
	<li>Using the microscope Look Up Table (LUT) settings, adjust the offset such that there is no positive FAM-YVAD-FMK staining in the untreated cells. This process can also be performed using caspase-1 deficient macrophages. 
	NOTE: The offset has to be adjusted for each channel used in the experiment, but setting the correct offset is most critical for the FAM-YVAD-FMK channel and fluorescent antibody channels. The offset for the DNA channel is less critical. Once the offset has been set, do not adjust this setting for the rest of the experiment.</li>
	<li>Switch the slide to the nigericin treated sample. Find a field that contains both cells that are positive and negative for FAM-YVAD-FMK staining. Adjust the imaging plane of the FAM-YVAD-FMK channel to the plane that has the highest intensity of positive staining. This will normally be the plane where the center of the inflammasome focus is imaged.</li>
	<li>Adjust the gain such that foci are visible in cells that have condensed nuclei. After setting both gain and offset, leave these settings for the rest of the imaging session. 
	NOTE: One easy way to determine if a cell is pyroptotic is by looking at nuclear morphology. Cells with active caspase-1 have rounded and condensed nuclei, while nucleoli are visible in cells without caspase-1 activation and the nuclear morphology is similar to those of untreated cells.</li>
	<li>Collect images of 5 randomly selected fields at 100X total magnification. This will normally result in about 40 cells per image. Repeat this process for each sample.</li>
	<li>Count the number of cells in each image using image analysis software. Then count the number of cells that have positive FAM-YVAD-FMK staining. Represent the level of caspase-1 activation as the percentage of FAM-YVAD-FMK positive cells.</li>
</ol>
7. LDH Release Assay
NOTE: For the LDH release assay, seed and prime the cells as described in the previous section, except use a 96 well plate. Do not remove the medium after priming.
<ol>
	<li>Add 50 &#956;L of DMEM-5 containing 10 &#956;M nigericin to the experimental wells and 50 &#956;L DMEM-5 to the spontaneous and 100% lysis control wells. Do not add glycine in this experiment since the goal is to measure cell lysis. Incubate for 60 min at 37 &#176;C and 5% CO2.</li>
	<li>After 30 min, add 10 &#956;L of 10x lysis buffer to the 100% lysis control wells and add 10 &#956;L of DMEM-5 to the other wells. During this incubation, remove 1 vial of substrate mix (1.5 mL of freeze-dried substrate) and 1 aliquot of assay buffer (~12 mL) from the freezer and let them thaw protected from light to room temperature. 
	NOTE: The substrate mix contains a tetrazolium salt (iodonitro-tetrazolium violet), which is converted to a red formazan product.</li>
	<li>After the 60 min total incubation, centrifuge the plate for 5 min at 500 x g at 10 &#176;C. Transfer 50 &#956;L of the supernatant to a clear flat-bottom plate. During this time, add 12 mL of assay buffer to the vial of substrate mix and incubate at room temperature for 5 min before using.</li>
	<li>Add 50 &#956;L of substrate to each well and incubate for 30 min in the dark. Include medium only wells for blank values. Check the plate after 15 min to make sure that the signal will not exceed the detection limit of the plate reader.</li>
	<li>After 30 min, add 50 &#956;L of stop solution (1 M acetic acid) and measure OD490 using a plate reader.</li>
	<li>Calculate the percentage of cell lysis using the following formula: 
	% Cytoxicity = ((Experimental - Spontaneous)/(Maximum - Spontaneous))&#215;100 
	Experimental: nigericin treated cells; spontaneous: untreated cells; maximum: cells exposed to lysis buffer</li>
</ol>

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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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-1-dependent+pore+formation+during+pyroptosis+leads+to+osmotic+lysis+of+infected+host+macrophages.">Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages.</a> Cell Microbiol. 8 (11), 1812-1825 (2006).</li><li>Bergsbaken, T., Fink, S. L., den Hartigh, A. B., Loomis, W. P., 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. 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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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The authors have no conflicts of interest to disclose

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 ...

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Research

JoVE Journal

Immunology and Infection

10.5K Views.  University of Washington. 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.

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

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...