an in vitro technique to study inflammasome complex formation in macrophages

An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

For immunofluorochemical analysis of the nigericin-treated cells, wash the probe-labeled cells, and incubate the cells in 250 microliters of fixation and permeabilization solution per well for 30 minutes on ice, protected from light.
Wash the macrophages three more times with fresh wash buffer, and label the cells with 250 microliters of primary antibody against apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain, or ASC, per well for one hour on ice.
Wash the cells at the end of the incubation, and label the samples with 250 microliters of the appropriate fluorescent dye-conjugated secondary antibody for another hour on ice, adding 4 microliters of an appropriate fluorescent nuclear staining dye during the last 5 minutes.
After washing the cells 3 times in cold wash buffer, mount coverslips onto the slides with 7 microliters of anti-fade mounting medium as demonstrated, and image the macrophages by fluorescence confocal microscopy.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Differentiation of Bone Marrow-derived Macrophages
<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.), 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 on day 3 or 4. 
	NOTE: It is important not to use tissue culture-treated plates, as macrophages will be difficult to detach and harvest.</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 washing 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>
2. 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 priming. 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>
3. Activation of the NLRP3 Inflammasome with Nigericin
<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 activation. 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>After 60 min, remove the medium and wash the cells three times for 5 min each with 1 mL of cold PBS.</li>
</ol>
4. Antibody Staining
NOTE: To label the cells with antibodies to detect ASC, seed, prime and expose the cells to nigericin identical to the previous section. The processing afterward is as follows:
<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 cover 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 a 7 &#956;L anti-fade mounting medium. Let the mounting medium harden overnight 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 the far-red fluorescent nucleic acid stain.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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)</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. Proprietary formulations. Contains 4.2% formaldehyde</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</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</td>
		</tr>
		<tr>
			<td>C57BL/6J mouse</td>
			<td>Jackson Laboratoy</td>
			<td>664</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>
	</tbody>
</table>

Source: den Hartigh, A. B., et al. <a href="https://app.jove.com/v/57463">Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.</a> J. Vis. Exp. (2018).
This video demonstrates a technique to study NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome formation in primary macrophages via immunofluorescence. The macrophages are first primed with bacterial lipopolysaccharide (LPS) to upregulate NLRP3 protein expression. Exposing the macrophages to nigericin&#8212;a potassium ionophore&#8212;leads to the activation of NLRP3 and the formation of inflammasomes, which are visualized via immunofluorescence.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a plate containing a culture of primary macrophages.
Introduce bacterial lipopolysaccharide, LPS, that binds to toll-like receptors on macrophages, triggering upregulation of the protein NLR family pyrin domain-containing 3 or NLRP3.
Add an ionophore that mediates the efflux of potassium ions. Decreased cytosolic potassium activates NLRP3. Activated NLRP3 oligomerizes and induces the recruitment of an adaptor protein, which in turn recruits pro-caspase-1, forming an inflammasome.
Auto-proteolysis converts pro-caspase-1 to active caspase-1, which mediates inflammatory cell death or pyroptosis. DNA damage during pyroptosis leads to nuclear condensation.
Fix the cells and treat with a detergent to permeabilize them.
Add a primary antibody binding to the inflammasome-associated adaptor protein, and a fluorescently-labeled secondary antibody that binds to the primary antibody, labeling the inflammasome.
Introduce a fluorescent nucleic acid stain to label the nucleus. Under a fluorescence microscope, cells display condensed nuclei &#8212; indicating pyroptosis &#8212; and foci of adaptor proteins &#8212; indicating inflammasome formation.

27 조회수. 대식세포에서 인플라마솜 복합체 형성을 연구하기 위한 시험관 내 기술

비디오: 대식세포에서 인플라마솜 복합체 형성을 연구하기 위한 시험관 내 기술 - 실험

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

Caspase 활성화 분자 형광 보완성을 사용 하 여 경로를 조명

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

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JoVE Journal

생화학

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.

인간 단핵구 유래 대식세포에서 근접성을 유도한 염증성 카스파제의 시각화

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 ...

면역학 및 감염병학

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

선천면역세포사멸을 평가하기 위한 카스파아제 활성화 평가

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

내레이션 대본

An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages