We acknowledge the Wilhelm Sander-Foundation (2017.008.02), the Center of Dynamic Systems (CDS), funded by the EU-program ERDF (European Regional Development Fund) and the DFG (LA 2386) for supporting our work. We thank Karina Guttek for supporting our experiments. We acknowledge Prof. Dirk Reinhold (OvGU, Magdeburg) for providing us primary T cells.

&#8203;T cell experiments were performed according to the ethical agreement 42502-2-1273 Uni MD.
1. Preparing cells for the experiment
NOTE: The average number of cells for this immunoprecipitation is 1 &#215; 107. Adherent cells have to be seeded one day before the experiment so that there are 1 &#215; 107 cells&#160;on the day of the experiment.
<ol>
	<li>Preparing adherent cells for the experiment
	<ol>
		<li>Seed 5-8 &#215; 106 adherent cells in 20 mL of medium (see the Table of Materials for the composition) for each condition in 14.5 cm dishes one day before the experiment starts.</li>
		<li>On the day of the experiment, ensure that the cells are 80-90% confluent and adherent to the dish. Discard the medium and add fresh medium to the adherent cells.</li>
	</ol>
	</li>
	<li>Preparing suspension cells for the experiment
	<ol>
		<li>Carefully place 1 &#215; 107 suspension cells in 10 mL of culture medium (see the Table of Materials for the composition) per condition in 14.5 cm dishes immediately before the experiment starts.</li>
		<li>If using primary cells, isolate primary T cells according to the previously described procedure21. Treat primary T cells with 1 &#181;g/mL phytohemagglutinin for 24 h, followed by 25 U/mL IL2 treatment for 6 days.</li>
		<li>Carefully place 1 &#215; 108 primary T cells in 10 mL of culture medium (see the Table of Materials for the composition) per condition in 14.5 cm dishes immediately before the experiment starts. 
		&#8203;NOTE: This higher number of primary T cells is recommended, as these cells are smaller.</li>
	</ol>
	</li>
</ol>
2. CD95L stimulation
<ol>
	<li>Stimulate the cells with CD95L (produced as described previously20 or commercially available (see the Table of Materials)). 
	&#8203;NOTE: The concentration of the CD95L and the time of stimulation are cell-type dependent13,15,22,23,24,25. Prepare one stimulation condition twice to generate a &#39;bead control&#39; sample in parallel.
	<ol>
		<li>Stimulate adherent cells with the selected concentration of CD95L. Hold the plate at an angle and pipet the ligand into the medium without touching the adherent cells.</li>
		<li>Stimulate suspension cells with CD95L by pipetting the ligand solution into the cell suspension.</li>
	</ol>
	</li>
</ol>
3. Cell harvest and lysis
<ol>
	<li>Place the cell dishes on ice. 
	NOTE: Do not discard the medium. Dying cells float in the medium and are important for the analysis.</li>
	<li>Add 10 mL of cold phosphate-buffered saline (PBS) to the cell suspension and scrape the attached cells off the plate. Collect the cell suspension in a 50 mL tube.</li>
	<li>Wash the cell dish with 10 mL of cold PBS twice and place the wash solution into the same 50 mL tube. Centrifuge the cell suspension at 500 &#215; g for 5 min, 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the cell pellet with 1 mL of cold PBS. Transfer the cell suspension into a 1.5 mL tube.</li>
	<li>Centrifuge the cell suspension at 500 &#215; g for 5 min, 4 &#176;C. Discard the supernatant and resuspend the cell pellet with 1 mL of cold PBS.</li>
	<li>Centrifuge the cell suspension at 500 &#215; g for 5 min, 4 &#176;C. Discard the supernatant and resuspend the cell pellet with 1 mL of lysis buffer (containing 4% protease inhibitor cocktail). Incubate it for 30 min on ice.</li>
	<li>Centrifuge the lysate at maximal speed (~17,000 &#215; g) for 15 min, 4 &#176;C.</li>
	<li>Transfer the supernatant (lysate) to a clean tube. Discard the pellet. Take 50 &#181;L of the lysate in another tube. Analyze the protein concentration by Bradford assay and take the amount of lysate corresponding to 25 &#181;g of protein in a vial. Add loading buffer (see the Table of Materials for the composition) to the vial. Store it at -20 &#176;C as lysate control.</li>
</ol>
4. Immunoprecipitation (IP)
<ol>
	<li>Add 2 &#181;L of anti-APO-1 antibodies and 10 &#181;L of protein A sepharose beads (prepared as recommended by the manufacturer) to the lysate. Add only 10 &#181;L of the beads to a separate tube containing lysate (stimulated sample) to generate a &#39;bead control.&#39; 
	NOTE: Use pipet tips with wide orifices either by cutting the tips or buying special tips for IP while handling the protein A sepharose beads.</li>
	<li>Incubate the mixture of lysate with antibodies/protein A sepharose beads with gentle mixing overnight at 4 &#176;C. Centrifuge the lysates with antibodies/protein A sepharose beads at 500 &#215; g for 4 min, 4 &#176;C. Discard the supernatant, add 1 mL of cold PBS to the beads, and repeat this step at least three times.</li>
	<li>Discard the supernatant. Aspirate the beads preferably with a 50 &#181;L Hamilton syringe.</li>
</ol>
5. Western blot
<ol>
	<li>Add 20 &#181;L of 4x loading buffer (see the Table of Materials for the composition) to the beads and heat at 95 &#176;C for 10 min. Heat the lysate controls at 95 &#176;C for 5 min.</li>
	<li>Load the lysates, IPs, and a protein standard onto a 12.5% sodium dodecyl sulfate (SDS) gel (see the Table of Materials for the gel preparation) and run with a constant voltage of 80 V.</li>
	<li>Transfer the proteins from the SDS gel to a nitrocellulose membrane. 
	NOTE: Here, the semi-dry technique, optimized for the proteins of interest, was used for the transfer over 12 min (25 V; 2.5 A= constant). Soak the nitrocellulose membrane and two mini-size transfer stacks in electrophoresis buffer (see the Table of Materials for the composition, prepare according to the manufacturer&#39;s instructions) for a few minutes before western blotting.</li>
	<li>Place the blotted membrane in a box and block it for 1 h in blocking solution (0.1% Tween-20 in PBS (PBST) + 5% milk). Incubate the membrane with the blocking solution under gentle agitation.</li>
	<li>Wash the membrane three times with PBST for 5 min each wash.</li>
</ol>
6. Western blot detection
<ol>
	<li>Add the first primary antibody at the indicated dilution (see the Table of Materials) to the membrane and incubate it overnight at 4 &#176;C with gentle agitation.</li>
	<li>Wash the membrane three times with PBST for 5 min each wash.</li>
	<li>Incubate the membrane with 20 mL of secondary antibody (diluted 1:10,000 in PBST + 5% milk) with gentle shaking for 1 h at room temperature.</li>
	<li>Wash the membrane three times with PBST for 5 min each wash.</li>
	<li>Discard PBST and add approximately 1 mL of horseradish peroxidase substrate to the membrane.</li>
	<li>Detect the chemoluminescent signal (see the Table of Materials). 
	NOTE: The exposure time and the number of captured images depend on the amount of protein in the cell and the specificity of the antibodies used. It must be established empirically for each antibody used for the detection.</li>
</ol>

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The authors have no conflicts of interest to disclose.

This approach was first described by Kischkel et al.27 and has successfully been developed since then by several groups. Several important issues have to be considered for efficient DISC-immunoprecipitation and monitoring caspase-8 processing in this complex.
First, it is essential to follow all washing steps during immunoprecipitation. Especially important are the final washing steps of the sepharose beads and the drying of the sepharose beads. This must be done correc...

One of the best-studied death receptors (DRs) is CD95 (Fas, APO-1). The extrinsic apoptotic pathway starts with the interaction of the DR with its cognate ligand, i.e., CD95L interacts with CD95 or TRAIL binds to TRAIL-Rs. This results in the formation of the DISC at the corresponding DR. DISC consists of CD95, FADD, procaspase-8/-10, and c-FLIP proteins1,2. Furthermore, the DISC is assembled by interactions between death domain (DD)-containing proteins, such as CD95 and FADD, and DED-containing proteins such as FADD, procaspase-8/-10, and c-FLIP (Figure 1). Procaspase-8 undergoes oligomerization via association of its DEDs, resulting in the formation of DED filaments, followed by procaspase-8 activation and processing. This triggers a caspase cascade, which leads to cell death (Figure 1)3,4. Thus, procaspase-8 is a central initiator caspase of the extrinsic apoptosis pathway mediated by CD95 or the TRAIL-Rs, activated at the corresponding macromolecular platform, DISC.
Two isoforms of procaspase-8, namely procaspase-8a (p55) and -8b (p53), are known to be recruited to the DISC5. Both isoforms comprise two DEDs. DED1 and DED2 are located at the N-terminal part of procaspase-8a/b followed by the catalytic p18 and p10 domains. Detailed cryo-electron microscopy (cryo-EM) analysis of procaspase-8 DEDs revealed the assembly of procaspase-8 proteins into filamentous structures called DED filaments4,6. Remarkably, the linear procaspase-8 chains were initially suggested to be engaged in the dimerization followed by procaspase-8 activation at the DISC. Now, it is known that those chains are only a substructure of the procaspase-8 DED filament, the latter comprising three chains assembled into a triple helix3,4,6,7.
Upon dimerization at the DED filament, conformational changes in procaspase-8a/b lead to the formation of the active center of procaspase-8 and its activation3,8. This is followed by procaspase-8 processing, which is mediated via two pathways: the first one goes via the generation of a p43/p41 cleavage product and the second one via the initial generation of a p30 cleavage product. The p43/p41 pathway is initiated by the cleavage of procaspase-8a/b at Asp374, resulting in p43/p41 and p12 cleavage products (Figure 2). Further, these fragments are auto-catalytically cleaved at Asp384 and Asp210/216, giving rise to the formation of the active caspase-8 heterotetramer, p102/p1829,10,11. In addition, it was shown that in parallel to the p43/p41 pathway of processing, procaspase-8a/b is also cleaved at Asp216, which leads to the formation of the C-terminal cleavage product p30, followed by its proteolysis to p10 and p1810 (Figure 2).
Procaspase-8a/b activation at the DED filament is strictly regulated by proteins named c-FLIPs12. The c-FLIP proteins occur in three isoforms: c-FLIPLong (c-FLIPL), c-FLIPShort (c-FLIPS), and c-FLIPRaji (c-FLIPR). All three isoforms contain two DEDs in their N-terminal region. c-FLIPL also has a C-terminal catalytically inactive caspase-like domain12,13. Both short isoforms of c-FLIP-c-FLIPS and c-FLIPR-act in an anti-apoptotic manner by disrupting DED filament formation at the DISC6,14,15. In addition, c-FLIPL can regulate caspase-8 activation in a concentration-dependent manner. This can result in both pro- and anti-apoptotic effects16,17,18. By forming the catalytically active procaspase-8/c-FLIPL heterodimer, c-FLIPL leads to the stabilization of the active center of procaspase-8 and its activation. The pro- or anti-apoptotic function of c-FLIPL is directly dependent on its amount at the DED filaments and the subsequent amount of assembled procaspase-8/c-FLIPL heterodimers19. Low or intermediate concentrations of c-FLIPL at the DISC result in sufficient amounts of procaspase-8/c-FLIPL heterodimers at the DED filament, which supports the activation of caspase-8. In contrast, increased amounts of c-FLIPL directly lead to its anti-apoptotic effects at the DISC20.
Taken together, the activation and processing of procaspase-8a/b at the DISC is a highly regulated process involving several steps. This paper discusses the measurement of procaspase-8 processing directly at the DISC as well as the analysis of the composition of this complex. This will be presented using CD95 DISC as the exemplary DR complex.

<table><tbody><tr><td>12.5% SDS gel</td><td>self made</td><td></td><td>for two separating gels:&lt;br/&gt; 3.28 mL distilled H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 2.5 mL Tris; pH 8.8; 1.5 M&lt;br/&gt; 4.06 mL acrylamide&lt;br/&gt; 100 &amp;micro;L 10% SDS&lt;br/&gt; 100 &amp;micro;L 10% APS&lt;br/&gt; 7.5 &amp;micro;L TEMED&lt;br/&gt; &lt;br/&gt; for two collecting gels:&lt;br/&gt; 3.1 mL distilled H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 1.25 mL Tris; pH 6.8; 1.5 M&lt;br/&gt; 0.5 mL acrylamide&lt;br/&gt; 50 &amp;micro;L 10% SDS&lt;br/&gt; 25 &amp;micro;L 10% APS&lt;br/&gt; 7.5 &amp;micro;L TEMED</td></tr><tr><td>14.5 cm cell dishes</td><td>Greiner</td><td>639160</td><td></td></tr><tr><td>acrylamide</td><td>Carl Roth</td><td>A124.1</td><td></td></tr><tr><td>Aerosol filter pipet tips with high recovery filter and wide orifice</td><td>VWR</td><td>46620-664 (North America) or 732-0559 (Europe)</td><td>Used for pipetting beads to the lysate</td></tr><tr><td>anti-actin Ab</td><td>Sigma Aldrich</td><td>A2103</td><td>dilution: 1:4000 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>anti-APO-1 Ab</td><td>provided in these experiments by Prof. P. Krammer or can be purchased by Enzo</td><td>ALX-805-038-C100</td><td>used only for immunoprecipitation</td></tr><tr><td>anti-caspase-10 Ab</td><td>Biozol</td><td>MBL-M059-3</td><td>dilution: 1:1000 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>anti-caspase-3 Ab</td><td>cell signaling</td><td>9662 S</td><td>dilution: 1:2000 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>anti-caspase-8 Ab C15</td><td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td><td>ALX-804-242-C100</td><td>dilution: 1:20 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>anti-CD95 Ab</td><td>Santa Cruz</td><td>sc-715</td><td>dilution: 1:2000 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>anti-c-FLIP NF6 Ab</td><td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td><td>ALX-804-961-0100</td><td>dilution: 1:10 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>anti-FADD 1C4 Ab</td><td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td><td>ADI-AAM-212-E</td><td>dilution: 1:10 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>anti-PARP Ab</td><td>cell signaling</td><td>9542</td><td>dilution: 1:1000 in PBST + 1:100 NaN&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>APS</td><td>Carl Roth</td><td>9592.3</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Carl Roth</td><td>4227.2</td><td></td></tr><tr><td>Bradford solution&lt;br/&gt; Protein Assay Dye Reagent Concentrate 450ml</td><td>Bio Rad</td><td>500-0006</td><td>used according to manufacturer&#039;s instructions</td></tr><tr><td>CD95L</td><td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td><td>ALX-522-020-C005</td><td></td></tr><tr><td>chemoluminescence detector&lt;br/&gt; Chem Doc XRS+</td><td>Bio Rad</td><td></td><td></td></tr><tr><td>cOmplete Protese Inhibitor Cocktail (PIC)</td><td>Sigma Aldrich</td><td>11 836 145 001</td><td>prepared according to manufacturer&#039;s instructions</td></tr><tr><td>DPBS (10x) w/o Ca, Mg</td><td>PAN Biotech</td><td>P04-53500</td><td>dilution 1:10 with H&lt;sub&gt;2&lt;/sub&gt;O, storage in the fridge</td></tr><tr><td>eletrophoresis buffer</td><td>self made</td><td></td><td>10x electrophoresis buffer:&lt;br/&gt; 60.6 g Tris&lt;br/&gt; 288 g glycine&lt;br/&gt; 20 g SDS&lt;br/&gt; ad 2 L H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 1:10 dilution before usage</td></tr><tr><td>glycine</td><td>Carl Roth</td><td>3908.3</td><td></td></tr><tr><td>Goat Anti-Mouse IgG1 HRP</td><td>SouthernBiotech</td><td>1070-05</td><td>dilution 1:10.000 in PBST + 5% milk</td></tr><tr><td>Goat Anti-Mouse IgG2b HRP</td><td>SouthernBiotech</td><td>1090-05</td><td>dilution 1:10.000 in PBST + 5% milk</td></tr><tr><td>Goat Anti-Rabbit IgG-HRP</td><td>SouthernBiotech</td><td>4030-05</td><td>dilution 1:10.000 in PBST + 5% milk</td></tr><tr><td>Interleukin-2 Human(hIL-2)</td><td>Merckgroup/ Roche</td><td>11011456001</td><td>for activation of T cells</td></tr><tr><td>KCl</td><td>Carl Roth</td><td>6781.2</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth</td><td>3904.1</td><td></td></tr><tr><td>loading buffer&lt;br/&gt; 4x Laemmli Sample Buffer,10 mL</td><td>Bio Rad</td><td>161-0747</td><td>prepared according to manufacturer&#039;s instructions</td></tr><tr><td>Luminata Forte Western HRP substrate</td><td>Millipore</td><td>WBLUFO500</td><td></td></tr><tr><td>lysis buffer</td><td>self made</td><td></td><td>13.3 mL Tris-HCl; pH 7.4; 1.5 M&lt;br/&gt; 27.5 mL NaCl; 5 M&lt;br/&gt; 10 mL EDTA; 2 mM&lt;br/&gt; 100 mL Triton X-100&lt;br/&gt; add 960 mL H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>medium for adhaerent cells DMEM F12 (1:1) w stable Glutamine,&amp;nbsp; 2,438 g/L</td><td>PAN Biotech</td><td>P04-41154</td><td>adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium</td></tr><tr><td>medium for primary T cells</td><td>gibco by Life Technologie</td><td>21875034</td><td>adding 10% FCS and 1% Penicillin-Streptomycin to the medium</td></tr><tr><td>milk powder</td><td>Carl Roth</td><td>T145.4</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth</td><td>P030.3</td><td></td></tr><tr><td>NaCl</td><td>Carl Roth</td><td>3957.2</td><td></td></tr><tr><td>PBST</td><td>self made</td><td></td><td>20x PBST:&lt;br/&gt; 230 g NaCl&lt;br/&gt; 8 g KCl&lt;br/&gt; 56.8 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; 8 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; 20 mL Tween-20&lt;br/&gt; ad 2 L H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; dilution 1:20 before usage</td></tr><tr><td>PBST + 5% milk</td><td>self made</td><td></td><td>50 g milk powder + 1 L PBST</td></tr><tr><td>PHA</td><td>Thermo Fisher Scientific</td><td>R30852801</td><td>for activation of T Cells</td></tr><tr><td>Power Pac HC</td><td>Bio Rad</td><td></td><td></td></tr><tr><td>Precision Plus Protein Standard All Blue</td><td>Bio Rad</td><td>161-0373</td><td>use between 3-5 &amp;micro;L</td></tr><tr><td>Protein A Sepharose CL-4B beads</td><td>Novodirect/ Th.Geyer</td><td>GE 17-0780-01</td><td>affinity resin beads prepared according to manufacturer&#039;s instructions</td></tr><tr><td>scraper</td><td>VWR</td><td>734-2602</td><td></td></tr><tr><td>SDS</td><td>Carl Roth</td><td>4360.2</td><td></td></tr><tr><td>shaker</td><td>Heidolph</td><td></td><td></td></tr><tr><td>sodium azide</td><td>Carl Roth</td><td>K305.1</td><td></td></tr><tr><td>TEMED</td><td>Carl Roth</td><td>2367.3</td><td></td></tr><tr><td>Trans Blot Turbo mini-size transfer stacks</td><td>Bio Rad</td><td>170-4270</td><td>used according to manufacturer&#039;s instructions</td></tr><tr><td>TransBlot Turbo 5x Transfer Buffer</td><td>Bio Rad</td><td>10026938</td><td>prepared according to manufacturer&#039;s instructions</td></tr><tr><td>TransBlot Turbo Mini-size nictrocellulose membrane</td><td>Bio Rad</td><td>170-4270</td><td>used according to manufacturer&#039;s instructions</td></tr><tr><td>Trans-Blot-Turbo</td><td>Bio Rad</td><td></td><td></td></tr><tr><td>Tris</td><td>Chem Solute</td><td>8,08,51,000</td><td></td></tr><tr><td>Triton X-100</td><td>Carl Roth</td><td>3051.4</td><td></td></tr><tr><td>Tween-20</td><td>Pan Reac Appli Chem</td><td>A4974,1000</td><td></td></tr></tbody></table>

measuring composition of cd95 death-inducing signaling complex and processing of procaspase-8 in this complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

To analyze caspase-8 recruitment to the DISC and its processing at the CD95 DISC, this paper describes a classical workflow, which combines IP of the CD95 DISC with western blot analysis. This allows the detection of several key features of caspase-8 activation at the DISC: the assembly of the caspase-8-activating macromolecular platform, recruitment of procaspase-8 to the DISC, and the processing of this initiator caspase (Figure 1 and Figure 2). This workflow ...

Watch this Scientific Journal Video about Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex at JoVE.com

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

measuring-composition-cd95-death-inducing-signaling-complex

Research

JoVE Journal

Biochemistry

2.5K Views.  Otto von Guericke University Magdeburg. Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Video: Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

Chemistry

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.

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

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Cancer Research

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Neuroscience

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.

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

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 ...

Immunology and Infection

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 ...

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.

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

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Biology

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://app.jove.com/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

Chemiluminescencyjny test Western Blot do przetwarzania białek

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...