The authors would like to acknowledge Olivier Manches, Ph.D., Davor Frleta, Ph.D., and Meagan O&#8217;Brien, M.D. for their support and feedback. This research was supported by the National Institute of Allergy and Infectious Disease and completed with funding from NIH grants Ruth L. Kirschstein National Research Service Awards for Individual Predoctoral Fellowships (F31) to Promote Diversity in Health-Related Research (AI089030) and RO1 (AI081848).

Ethics Statement: Research samples are obtained and stored for research with donors&#8217; consent. All samples should be coded or anonymized prior to use. This protocol follows the guidelines of our Institutional Review Board.
1. Differentiation of Human Peripheral Blood Monocytes into Monocyte Derived Dendritic Cells.
Note: Human buffy coats serve as the source of human peripheral blood cells (PBMCs) and were obtained from the New York Blood Center (New York, NY). Blood donors are healthy volunteers. The 5 day procedure begins with the plating of human peripheral blood mononuclear cells (PBMCs) onto tissue culture flasks35,36. Notable differences from the published protocols are the following:
<ol>
	<li>Use 225 cm2 non pyrogenic polystyrene tissue culture flasks with a filter cap to adhere a total of 2x108 PBMCs per flask instead of 10 cm polystyrene tissue culture plates (58 cm2) (step 1).</li>
	<li>Prepare media with 5% pooled human serum (PHS; 30 ml) in 500 ml RPMI-1640 + L-glutamine, 5 ml 1 M HEPES buffer, and 1.4 ml 50 mg/ml Gentamicin (5% PHS media) followed by filtration through a 20 &#181;M Filter. Add a total of 50 ml 5% PHS media per 225 cm2 tissue culture flask.</li>
	<li>Wash adhered cells three times with 25 ml fresh RPMI-1640. Shake vigorously for 5 sec&#160;during each wash and aspirate non adherent cells (step 4).</li>
	<li>Add 190 &#181;l of 400 IU/&#181;l IL-4 and 380 &#181;l of 100 IU/&#181;l GM-CSF per flask (step 3) on day 0, 2, and 4. Day 0 is defined as the day PBMCs are initially plated in Step 1 (Step 8).</li>
	<li>Harvest day 5 moDCs at a concentration of 1x106 cells/ml (step 15).</li>
	<li>Use moDCs immediately in their resting state. Steps 17-22 are never performed. Note: Samples should be processed as soon as possible after collection for best results.</li>
	<li>Aliquot moDCs at a concentration of 2x105 cells/well (200 &#181;l/well of the 1x106 cells/ml) in a 96 well round bottomed plate (western blot, ELISA, FACs) or onto a poly-L-lysine treated chamberslide (microscopy) for experimentation. Note: There are at least 4 conditions: Completely unstimulated (negative control), priming only, activation only, and priming followed by activation. Conditions can be expanded to include diluent controls for R848 and nigericin if desired. If the downstream assay is ICS, duplicates are necessary for isotype controls.</li>
</ol>
2. Priming the Inflammasome - Signal 1
<ol>
	<li>Reconstitute lyophilized R848 in DMSO according to the manufacturer&#39;s instructions. Dilute working stock at RT with RPMI-1640.</li>
	<li>Add R848 at a 10 &#181;M final concentration to appropriate wells for 18 hr.&#160;Place cells in an incubator at 37 &#176;C and 5% CO2.</li>
</ol>
3. Activating the NLRP3 Inflammasome - Signal 2
<ol>
	<li>Reconstitute lyophilized nigericin in ethanol according to the manufacturer&#39;s instructions. Dilute working stock at RT with RPMI-1640 before adding to the appropriate conditions.</li>
	<li>Add nigericin at a 20 &#181;M final concentration and return to the incubator at 37 &#176;C and 5% CO2 for 6 hr. No washing steps occur between priming and during activation. Note: Secretion of IL-1&#946; is increased by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol, or carbonyl cyanide chlorophenylhydrazone37,38. Therefore, addition of these secretory inhibitors are not recommended when analyzing inflammasome priming or activity since it will alter secretion of IL-1&#946;.</li>
</ol>
4. IL-1&#223; Sample Collection
<ol>
	<li>Centrifuge the plate with cells and supernatants at 974 x g for 3 min.</li>
	<li>Without disturbing the cell pellet, aspirate the supernatant and transfer to a separate round bottomed plate in order to measure cytokine secretion from the supernatants. Note: Supernatants can be temporarily stored at -20 &#176;C or -80 &#176;C for long term storage.</li>
	<li>Wash the cellular pellets three times with 200 &#181;l of 1x PBS at 974 x g for 3 min&#160;to remove any extracellular IL-1&#946; from the cellular samples. Note: When washing cell pellets from a 96 well plate, remove the wash by swift plate inversion into a collection bin so as to not disturb the sample.</li>
</ol>
5. Measuring IL-1&#223; From Cellular and Supernatant Samples
<ol>
	<li>Intracellular Cytokine Staining (ICS): The ICS protocol is described below39,40.&#160;Note: do not suspend the cells if the downstream assay is microscopy. All washes and aspirations should be done without disturbing the cell layer/pellet (depending on the downstream assay).
	<ol>
		<li>Add 100 &#181;l&#160;of 5% PHS media into appropriate wells. Add appropriate volume (approximately 1 &#181;l/2x105 cells, or 1 &#181;l/well) of fluorescently labeled &#945;-CD11c and &#945;-CD14 (phenotype markers; clone B-ly6 and M&#966;P9, respectively) or isotype control to the cells for 10 min at RT in the dark.</li>
		<li>Wash three times with 1x PBS at 974 x g for 3 min.</li>
		<li>Fix the cells by adding 100 &#181;l of 4% PFA for 20 min&#160;at RT in the dark.</li>
		<li>Pause Point: Wash with 100 &#181;l 1x PBS at 974 x g for 3 min&#160;and store at 4 &#176;C O/N&#160;in 1x PBS.</li>
		<li>Add 100 &#181;l of permeabilization buffer to cells for 30 min. Note: permeabilization buffer is composed of 1x PBS with 0.3% Triton X-100, and 1% bovine serum albumin (BSA) for permeabilization. Do not disturb adherent cells when downstream assay is microscopy.</li>
		<li>Add appropriate volume (approximately 62 ng antibody/2x105 cells, approximately or 2.5 &#181;l/well) of &#945;-IL-1&#946;-FITC (clone 8516) or isotype control for 2 hr&#160;in an incubator at 37 &#176;C.</li>
		<li>Wash cells three times with 200 &#181;l of permeabilization buffer at 974 x g for 3 min&#160;in the dark.</li>
		<li>Optional: Stain with DAPI, add mountant, and place coverslip gently on top of a glass slide. Allow mountant to cure O/N&#160;before capturing microscopy images.</li>
		<li>Acquire the data by FACs or microscopy. Note: Compare the isotype for each condition to the appropriate staining when analyzing by FACs or microscopy.
		<ol>
			<li>FACs: Acquire one sample at a time. Set the FSC/SSC gate on the live cells, followed by gating on CD11c+CD14- cells before analyzing the moDC population pro-IL-1&#946; staining.</li>
			<li>Microscopy: Set the exposure time using the positive staining sample - R848 treated. Use DAPI+pro-IL-1&#946;+ and DAPI+pro-IL-1&#946;- to determine percentage of pro-IL-1&#946;+ expressing moDCs.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>SDS-Page: Immunoblotting is performed to detect pro-IL-1&#946;. Note: The technique described below combines standard immunoblotting technique, gradient 4-20% polyacrylamide gel, and fluorescent or chemiluminescent detection41,42 .
	<ol>
		<li>Lyse cells directly in 10 &#181;l denaturing lysis buffer (5 &#181;l &#946;-mercaptoethanol/950 &#181;l Laemmli sample buffer followed by diluting 1:1 with cell lysis buffer). Transfer lysate to 1.5 ml Eppendorf tubes.</li>
		<li>Heat lysate on a dry plate for 10 min&#160;at 100 &#176;C.</li>
		<li>Spin lysate at 20,800 x g on a minicentrifuge for 1 min to concentrate the liquid volume. Pause point: samples can be frozen at -20 &#176;C for short term storage.</li>
		<li>Load the total volume onto the polyacrylamide gel. Run 140 V through the gel box for approximately 1 hr, or until the dye front runs off the bottom of the gel.</li>
		<li>Transfer the protein from the polyacrylamide gel onto a PVDF Immobilon-FL membrane for 90 min at 100 V. Block the membrane with 5% BSA in Tris Buffered Saline/ 0.1% Tween-20 (TBS-T) for 1 hr. Note: PVDF Immobilon-FL Is best for use with fluorescent detection. A membrane with a different composition is recommended for other detection techniques to increase the signal to background ratio.</li>
		<li>Dilute primary and secondary antibodies in 10 ml of 5% BSA/TBS-T. Perform primary antibody incubations at 4 &#176;C O/N while shaking and secondary antibody at RT for 1 hr while shaking.</li>
		<li>Wash the membrane 3 times with TBS-T for 5 min while shaking between primary and secondary antibody incubations and again between secondary incubations and imaging. Note: Bands can be detected using fluorescent or chemiluminescent detection. Follow the manufacturer&#8217;s instructions for detection.</li>
	</ol>
	</li>
	<li>ELISA: Samples that are frozen for storage need to equilibrate to RT before analysis. Spin down samples in a centrifuge to consolidate supernatant condensation. Follow the manufacturer&#8217;s instructions for measurement of IL-1&#946;. Note: In this protocol IL-1&#946; is specifically measured; however, simultaneous measurement of TNF&#945;, IL-6, and the immunomodulatory cytokine IL-10 ensure the appropriate conditions were primed.</li>
</ol>

The authors have no conflicts of interest to disclose.

Inflammatory cytokines are integral in steering the innate and adaptive immune response to fight viral infection. Secreted IL-1&#946; has been shown to increase during Influenza infection3,43,44 . The precise mechanisms by which these cytokines are processed in response to viral recognition in human dendritic cells are not fully understood. Myeloid DC isolation kits are expensive and time consuming. Isolation kits and FAC sorting may unintentionally stress or activat...

Activation of innate immune system is required to steer adaptive immune responses during infection, disease, and vaccination7. Dendritic cells are the most potent antigen presenting cells of the innate immune system; they are specialized for uptake of antigens, migration to lymph nodes, and activation of na&#239;ve CD4+ and cytolytic CD8+ T-cells8-10. To enable rapid pathogen detection the innate immune system utilizes numerous germline encoded pattern recognition receptors (PRR) that recognize conserved pathogen derived motifs or host derived markers of cell stress and damage. Toll like receptors (TLRs) are membrane bound pattern recognition receptors that recognize certain extracellular phagocytized pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). By contrast nod like receptors (NLRs) are cytosolic and respond to a diverse range of PAMPs and DAMPs. Nod like receptors represent a second line of defense against pathogens that evade cell surface and endocytic PRRs. The interaction of pathogen derived, or &#8220;danger&#8221; associated, factors with TLR and NLR ligands leads to a state of DC maturation resulting in increased DC interaction with other immune cells and promotion of T cell and natural killer cell activation11.
Interleukin-1&#946; is a crucial component of the host defense against infection. Upon recognition of a microorganism, the highly proinflammatory cytokine, IL-1&#946;, is secreted and functions as a chemo attractant and activator of innate and adaptive immune cells. In vivo IL-1&#946; is largely responsible for the acute phase response including fever and inflammatory cytokine synthesis12.
Most NLRs contain a C terminal leucine rich repeat domain that is thought to function in ligand sensing, a central nucleotide binding domain (NACHT) that is important for NLRP3 oligomerization, and an N terminal effector domain (PYD in NLRP3) that mediates signal transduction to downstream targets through protein protein interactions. The NLRP3 protein defines the most intensely studied inflammasome complex. This protein is a member of the NLR family and has the ability to form a multi molecular protein complex composed of NLRP3, the adaptor protein PYCARD (also known as ASC), and ICE. Upon inflammasome activation PYCARD binds to NLRP3 N terminal domains and recruits ICE via caspase activation and recruitment domain (CARD) domains. Interleukin-1 converting enzyme is initially generated as a zymogen containing a CARD motif at its N-terminus. Inflammasome formation results in bringing two ICE molecules sufficiently close to induce their autocatalytic activation. The inflammasome complex is necessary for activating ICE thus allowing it to convert cytoplasmic pro-IL-1&#946; to mature cytokine.
Successful secretion of IL-1&#946; in DCs requires sensing of two different and independent danger signals. First, TLR sensing of PAMPs, DAMPs, or cytokine signaling (TNF&#945; or IL-1&#946;) causes an upregulation of cytoplasmic pro-IL-1&#946; protein expression. A second, often different, signal is required for inflammasome complex formation upstream of ICE maturation. A few inflammasome stimulating signals include bacterial membrane pore forming toxins (such as nigericin), lysosomal disrupting crystals (such as monosodium urate crystals, MSU), and extracellular ATP. The upstream mechanism leading to NLRP3 inflammasome activation by these diverse activators is unclear. Studies investigating signaling upstream of inflammasome formation proposes that intracellular events, such as induction of hypokalemia or reactive oxygen species (ROS) indirectly activate the inflammasome13-28.
Amongst the different viral activators of the NLRP3 inflammasome is influenza, which provides both the primary and secondary signal required for IL-1&#946; secretion3,29-33 . Using mouse NLRP3 knockout models it was found that IL-1&#946; secretion in DCs is NLRP3 dependent32. Additionally, NLRP3 knockout mice attracted fewer leukocytes to the site of infection and experienced higher mortality2,5. Two recent papers suggest a mechanism for NLRP3 inflammasome activation during Influenza virus infection; first, priming through recognition of viral RNA by TLR7 or TLR8 (depending on TLR expression of the responding cell) or through sensing of commensal bacteria by other TLRs to induce cytoplasmic pro-IL-1&#946; expression, followed by a second signal, activation of NLRP3 inflammasome formation by viral ion channel protein M2 on the trans&#160;Golgi network33,34. In the latter step, triggering of the NLRP3 inflammasome is accomplished by disturbance of the intracellular ionic milieu leading to ROS production, which is, simply, sensed by NLPR3 as a signal to form the inflammasome. However, the precise mechanism of inflammasome activation upstream of ICE activity during Influenza infection still remains unclear.
This work describes a technique valuable for studying the NLRP3 inflammasome in human moDCs that can be used as a foundation for further investigation of the pathway underlying DC based IL-1&#946; secretion in response to TLR8 ligation with R848 followed by activation of the inflammasome by a well known activator of NLRP3, nigericin. Variations of this method can be used with other cell types including, but not limited to: monocytes, macrophages, other DC subsets, and epithelial cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:1081px;" width="1081">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Name of Reagent/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:121px;">Catalog Number</td>
			<td style="width:391px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">IL-4</td>
			<td style="width:179px;">R&#38;D</td>
			<td style="width:121px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">GM-CSF</td>
			<td style="width:179px;">Genzyme</td>
			<td style="width:121px;">NDC 58468-0180-2</td>
			<td>We acquire this item through our local pharmacy with a prescription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">RPMI 1640 with L-glutamine</td>
			<td style="width:179px;">Cellgro</td>
			<td style="width:121px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Peripheral blood mononuclear cells</td>
			<td style="width:179px;">New York Blood Center</td>
			<td style="width:121px;"></td>
			<td>PBMCs were isolated from the blood of healthy donors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">12-well tissue culture plates</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:121px;">3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">96-well round bottom tissue culture plates</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:121px;">3799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-IL-1&#946;-FITC</td>
			<td style="width:179px;">R&#38;D</td>
			<td style="width:121px;">IC201F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">FITC isotype control</td>
			<td style="width:179px;">Miltenyi Biotec</td>
			<td style="width:121px;">130-092-213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-&#946;-Tubulin</td>
			<td style="width:179px;">Santa Cruz</td>
			<td style="width:121px;">SC-9014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-IL-1&#946;</td>
			<td style="width:179px;">R&#38;D</td>
			<td style="width:121px;">mab201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">PVDF Immobilon-FL membrane</td>
			<td style="width:179px;">Millipore</td>
			<td style="width:121px;">IPFL00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">gradient 4-12 % polycrylamide gel</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">161-1159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">laemmli sample buffer</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">BSA</td>
			<td style="width:179px;">Equitech Bio Inc</td>
			<td style="width:121px;"></td>
			<td>30% solution sterile/filtered</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">PFA</td>
			<td style="width:179px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">15710</td>
			<td>16% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">human inflammatory cytokine bead array kit</td>
			<td style="width:179px;">BD</td>
			<td style="width:121px;">551811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">nigericin</td>
			<td style="width:179px;">Invivogen</td>
			<td>tlrl-nig</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">R848</td>
			<td>3M Corp.</td>
			<td style="width:121px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-CD14</td>
			<td style="width:179px;">BD</td>
			<td style="width:121px;">340436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#945;-CD11c</td>
			<td style="width:179px;">BD</td>
			<td style="width:121px;">555392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">&#946;-mercaptoethanol</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td>M6250-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">TBS</td>
			<td style="width:179px;"></td>
			<td style="width:121px;"></td>
			<td>On site stock room</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Tween-20</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:121px;">P2287-100mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"><a>Nunc EasYFlask 225cm2, Filter Cap, 70mL working volume, 30/Cs</a></td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:121px;">159933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">20 &#956;M Sterile Disposable Filter Units</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:121px;">569-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gentamicin</td>
			<td style="width:179px;">Invitrogen</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hepes</td>
			<td style="width:179px;">Invitrogen</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">goat &#945;-mouse IRDye 800CW</td>
			<td style="width:179px;">Licor</td>
			<td style="width:121px;">926-32210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">donkey &#945;-rabbit IRDye 680RD</td>
			<td style="width:179px;">Licor</td>
			<td style="width:121px;">926-68073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Spectra multicolor broad range protein ladder</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:121px;">26634</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Tris Glycine SDS 10x</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">1610732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Tris Glycine 10x</td>
			<td style="width:179px;">Bio Rad</td>
			<td style="width:121px;">161-0734</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Methanol - 4L</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:121px;">A433P-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Prolong Gold antifade Reagent with DAPI</td>
			<td style="width:179px;">Life Technologies</td>
			<td>P-36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">8 chamber polystyrene vessel tissue culture treated glass slide</td>
			<td style="width:179px;">BD Falcon</td>
			<td style="width:121px;">354108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Poly-L-Lysine</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:121px;">P4707</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

These techniques measure TLR8 priming with R848. Intracellular cytokine staining for pro-IL-1&#946; allows for microscopy and FACs readouts from CD14-CD11c+ moDCs. Both techniques can be quantified relative to a non primed, or resting, cell control as well as an isotype control (Figures 1 and 2). Percent of pro-IL-1&#946;+ staining cells is multiplied by the geometric median of this population to provide the median fluorescent intensity (...

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

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.

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 ...

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JoVE Journal

Immunology and Infection

20.7K Views.  New York University School of Medicine. Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β 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-1β expression.

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

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

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.

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 ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...