eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulfonic acid (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&#160;et al.), put bone marrow cells in a 15 cm non-tissue culture-treated Petri dish (1.2-1.5 x 107&#160;cells). Incubate the plate at 37 &#176;C and 5% carbon dioxide, or CO2&#160;for 7 days. Add an additional 30 mL of differentiation medium to the plate on day 3 or 4.&#160;&#160; 
	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&#160;cells/mL. For microscopy experiments, seed 1 mL of cells on coverslips in 24 well plates. For lactate dehydrogenase (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&#160;before starting the priming process.</li>
</ol>
3. Priming
<ol>
	<li>Replace the medium with fresh medium (DMEM-5) containing 100 ng/mL lipopolysaccharide or 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&#160;Salmonella minnesota&#160;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 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>During the last 45 minutes, 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 minutes, 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 a 7 &#956;L anti-fade mounting medium. Let the mounting medium harden overnight 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.</li>
</ol>
5. 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>

<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)&#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>FAM-YVAD-FMK</td>
			<td>Immunocytochemistry Technologies</td>
			<td>98</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>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>C57BL/6J mouse</td>
			<td>Jackson Laboratoy</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase-1/11-/- mouse</td>
			<td>Jackson Laboratory</td>
			<td>16621</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>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>

detection of inflammasome activation via fluorescence microscopy

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.&#160;(2018)
This video demonstrates a method to detect NLRP3 inflammasome activation in macrophages using fluorescent reporter probes. The cells are treated with potassium ionophores and glycine to induce inflammasome assembly and caspase-1 activation. Fluorescent reporter probes visualize activated caspase-1, and cells are stained with a fluorescent dye for nuclei visualization.

Detection of Inflammasome Activation via Fluorescence Microscopy

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

To detect NOD-like receptor P3, or NLRP3, inflammasome activation, take a multi-well plate containing activated macrophages expressing NLRP3 proteins.
Treat the cells with media containing ionophores and glycine. Ionophores induce potassium ion efflux, activating and oligomerizing NLRP3 proteins. Oligomerized NLRP3 recruits adaptor proteins, facilitating the binding of pro-caspase-1, forming the NLRP3 inflammasome complex.
On the inflammasome, proximity triggers pro-caspase-1 auto-cleavage, releasing active caspases, initiating pyroptosis and nuclear condensation. Additionally, glycine stabilizes cell structure, preventing cell lysis.
Treat macrophages with fluorescent reporter probes containing a caspase-1 binding group and a fluorophore linked via an amino acid sequence. The activated caspase-1 recognizes the probe&#39;s amino acid sequence and binds to its caspase-binding group, enabling its visualization.
Fix the cells and overlay them with fluorescent dye to stain the nuclei. Image under a fluorescence microscope.
Count macrophages with blue condensed nuclei and green fluorescent foci of activated caspase-1, suggesting NLRP3 inflammasome activation.

detection-of-inflammasome-activation-via-fluorescence-microscopy

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

120 Views. Fonte: den Hartigh, A. B. et al., Rilevamento dell'attivazione dell'inflammasoma e della morte delle cellule piroptotiche nei macrofagi derivati dal midollo osseo murino. J. Vis. Exp.(2018) Questo video mostra un metodo per rilevare l'attivazione dell'inflammasoma NLRP3 nei macrofagi utilizzando sonde reporter fluorescenti. Le cellule vengono trattate con ionofori di potassio e glicina per indurre l'assemblaggio dell'inflammasoma e l'attivazione della caspasi-1. Le sonde reporter fluorescenti v...

Video: Rilevamento dell'attivazione dell'inflammasoma tramite microscopia a fluorescenza - Concept

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

Misurazione dell'attività della caspasi mediante un saggio fluorometrico o citometria a flusso

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Ricerca

JoVE Journal

Biochimica

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Identificare le caspasi e i loro motivi che scindono le proteine durante l'infezione da virus dell'influenza A

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

Immunologia e infezioni

Quantification of Immunostained Caspase-9 in Retinal Tissue

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.

Detection of Inflammasome Activation via Fluorescence Microscopy

Trascrizione

Detection of Inflammasome Activation via Fluorescence Microscopy