1. Cloning and Cell Line Generation
<ol>
	<li>PCR amplify the NBB region, corresponding to amino acid residues 1-140 (NBB140), from human MLKL cDNA for in frame standard restriction enzyme-based cloning with the oligomerization domain 2x FK506 binding protein (2xFKBP or 2xFV) and Venus fluorescent protein into the Doxycycline (Dox)-inducible retroviral vector pRetroX-TRE3G to obtain NBB140-2xFV-Venus (Table 1, Figures 1A-B).</li>
	<li>Immortalize primary mlkl-/- or ripk3-/- mlkl-/-&#160;mouse embryonic fibroblasts (MEFs) obtained from the respective mice available in the Green laboratory by transient transfection with SV40-large antigen expressing plasmid. Select immortalized cells in ~1&#8211;2 weeks, and maintain in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and streptomycin, 1 mM sodium pyruvate, and nonessential amino acids at 37 &#176;C and 5% CO2 in standard tissue culture incubators.</li>
	<li>Transduce SV40-immortalized mlkl-/- MEFs with the reverse tetracycline controlled transactivator (rtTA)-containing retrovirus expressed from a plasmid containing the blasticidin resistance gene (our modified plasmid) and treat for 1 week with 5 &#956;g/mL blasticidin to select for cells stably expressing rtTA28.</li>
	<li>Transduce the rtTA-expressing MEFs with the Dox-inducible retroviral vector containing the chimeric MLKL (pRetroX-NBB140-2xFV-Venus-puro) and select using 4 &#956;g/mL puromycin for one week, 2 days after transduction.</li>
</ol>
2. Live-Cell Microscopy Imaging of MLKL-mediated Necroptosis
<ol>
	<li>Plate mlkl-/- MEFs expressing NBB140-2xFV-Venus at a density of 50,000 cells per well (1 mL/well) in 24 well plates, and incubate overnight (Figure 2A). Use live-cell staining for automated cell counting (see Table of Materials).</li>
	<li>Treat cells for 12 h with Dox (0.5 &#956;g/mL) to induce the expression of NBB140-2xFV-Venus.</li>
	<li>Induce necroptosis with 25 nM FKBP dimerizer (Dim) in addition to&#160;25 nM membrane-impermeable green fluorescent dye (see Table of Materials) in standard DMEM (see 1.2). Cells undergoing necroptosis accumulate the dye as their plasma membrane integrity is compromised by NBB140-mediated rupture. Perform assays in triplicate or quadruplicate.</li>
	<li>To monitor necroptosis using single- or dual-color imaging systems (see Table of Materials), place the vessels in the respective imaging unit housed in a mammalian tissue culture incubator.</li>
	<li>Open the software (see Table of Materials) and select the tab Schedule Upcoming Scans under the Tasks List. 
	NOTE: This protocol is for imaging using single-color systems, but a similar software is available for&#160;dual-color systems.</li>
	<li>Select the &#34;Tray, Vessel, Scan Types&#34;, and the &#34;Scan Pattern&#34; in the &#34;Physical Layout&#34; tab and &#34;Fast Fluorescence&#34; in the Properties tab.</li>
	<li>Add the &#34;Scan Time&#34; by clicking on the &#34;Timeline&#34; window and the total number of time points and frequency of acquisition (typically 1 acquisition every 0.5 h to 1 h for 6 h to 12 h, or every 5 min for fast-kinetics necroptosis) and start image acquisition by selecting &#34;Apply&#34; (red button).</li>
	<li>Quantify necroptosis using the image analysis software (see Table of Materials) by selecting from the &#34;Tasks Pane&#34;, &#34;Object Counting New Analysis with Fixed Segmentation Threshold above the background level&#34;, which can be determined by hovering the mouse cursor over background regions in several images used in the analysis.</li>
	<li>Examine fluorescent objects by selecting Previewing using current image and refine the Threshold levels as needed.</li>
	<li>Integrate green fluorescence counts by opening&#160;the &#34;Launch New Analysis&#34; tab, choose the &#34;Time Range&#34;, select the wells to be analyzed, enter the &#34;Analysis Job Name&#34;, and press &#34;OK&#34;.</li>
	<li>Access analyzed data from the &#34;Analysis Jobs&#34; tab by double-clicking on the respective job name and exporting from the Graph/Export window for additional analysis in external graphing software (see Table of Materials).</li>
	<li>Quantitate data as green fluorescence positive (+ve) counts/mm2 or normalize the counts by confluence and express the data as green fluorescence +ve counts/confluence.</li>
	<li>Optionally, at the experimental endpoint, stain cells with 100 nM of the membrane-permeant green fluorescent dye (see Table of Materials). Normalize data to % necroptotic cells as the ratio of green fluorescence/green fluorescence at endpoint.</li>
</ol>
3. Live-cell Confocal Microscopy Imaging of Plasma Membrane Recruitment and Permeabilization by MLKL
<ol>
	<li>Plate mlkl-/- MEFs expressing NBB140-2xFV-Venus at a density of 20,000 cells per well (0.5 mL/well) in a glass bottom 4-well microscopy chamber pretreated with 50 &#181;g/mL fibronectin&#160;in PBS at 37 &#176;C for 30 min and incubate for ~12 h (Figure 2B).</li>
	<li>Treat the cells with Dox (0.5 &#956;g/mL) for ~12 h to induce the expression of NBB140-2xFV-Venus.</li>
	<li>Induce necroptosis by incubating with fresh medium (1 mL/well) containing 25 nM Dim to induce NBB140-2xFV-Venus activation and translocation to the plasma membrane.</li>
	<li>Place the chamber on a spinning disc laser scanning confocal microscope built on an inverted stand equipped with environmental control and a 63 1.4 NA objective and begin imaging acquisition using software of choice (see Table of Materials).</li>
	<li>Initialize the software, select &#34;Focus&#34; icon and YFP filter configuration (excitation wavelength 515 nm).</li>
	<li>Locate the field of view and focal plane, and engage the focus maintenance system using the &#34;Definite Focus&#34; tab.</li>
	<li>To begin acquisition, select the &#34;Capture&#34; tab followed by assignment of filter configuration, appropriate EMCCD camera exposure time and intensification gain, and time-lapse acquisition parameters including interval and length of acquisition.</li>
	<li>Monitor cellular redistribution of the fluorescent NBB140-2xFV-Venus protein during necroptosis by acquiring images every 10 s by a EMCCD camera using a 515 nm laser (Movie 1). 
	NOTE: Photobleaching is a concern with fluorescent protein imaging. Venus is one of the more resistant fluorescent proteins to photobleaching29. If fluorescent proteins that are more susceptible to photobleaching are used, image every 30 s or longer to allow recovery of signal between imaging time points.</li>
	<li>To monitor plasma membrane association of NBB140-2xFV-Venus during necroptosis, image every 10 s the sample prepared in 3.3 by total internal reflection fluorescence (TIRF) microscopy using a microscope equipped with an automated TIRF slider and a 100X 1.4 NA objective and an EMCCD camera (Movie 2). Follow steps 3.5&#8211;3.7, but adjust the TIRF angle within the TIRF focus tab according to sample and bottom glass thickness of the microscopy chamber. 
	NOTE: Plasma membrane markers such as LCK-C-RFP may be used as controls for plasma membrane localization in TIRF analysis.</li>
	<li>Perform image processing to enhance quality by convolution with the negative normalized second derivative of a Gaussian function (Marr algorithm).</li>
</ol>
4. Electron Microscopy
<ol>
	<li>Plate mlkl-/- MEFs expressing NBB140-2xFV-Venus at a density of 5 million cells in a plasma-coated 150 mm cell culture dish and incubate for 12 h (Figure 3).</li>
	<li>Treat the cells with Doxycycline (0.5 &#956;g/mL) to induce the expression of NB1-140-2xFV-Venus for 12 h.</li>
	<li>Induce NBB140-2xFV-Venus activation and translocation to the plasma membrane with 25 nM of homodimerizer for 5 min. This time is established from the kinetics of necroptosis observed in live-cell imaging.</li>
	<li>Remove the media&#160;and fix cells using 10 mL of 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (cacodylate buffer) pre-warmed at 37 &#176;C.</li>
	<li>Collect the sample by scraping and spin the samples for 10 min at 500 x g. Discard the supernatant.</li>
	<li>Postfix the sample for 1.5 h in reduced 2% osmium tetroxide with 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate buffer. Osmium tetroxide is a staining agent widely used in TEM to provide contrast. We used TEM as preliminary analysis prior to SEM of cell undergoing necroptosis.</li>
	<li>Rinse the sample 5 times in ultrapure water for 5 min each at 500&#160;x g, followed by rinses in buffer (see 4.6), water, and ethanol on an automatic processor. Discard the supernatant.</li>
	<li>For the SEM, stain the previously fixed samples with 1% uranyl acetate and lead aspartate heavy-metal stain31.</li>
	<li>Dehydrate the samples through a graded series of alcohol and propylene oxide solutions.</li>
	<li>Embed the sample in hard resin32 to obtain a resin block.</li>
	<li>Cut thick sections of 0.5 &#956;m to determine the correct area of analysis.</li>
	<li>Coat the samples with an ultra-thin film of electrically-conducting metal (Iridium).</li>
	<li>Image and analyze the samples (Figure 3). For quantification, a low-magnification image of the resin block surface with exposed cells is scanned at 5 keV using an electron microscope.</li>
	<li>Visualize cells across individual images captured by SEM or imaging software may be utilized to join multiple fields-of-view into one contiguous image30. Roughly 100 cells may be used to evaluate the fraction of cells undergoing necroptosis in this manner by generating a high-resolution montage in software of choice (see Table of Materials). 
	NOTE: Scoring necrotic morphology by SEM compares normal cell features with the progression of pre-necrotic or necrotic phenotypes. These features include plasma membrane alterations including microvilli disappearance, flattening, ruptures in cells ranging from 10 nm to 1 &#181;m in size, or loss of organelle structure across at least 30&#8211;40% of the cytosolic cell area.</li>
</ol>
5. Lipid Binding of MLKL by Nuclear Magnetic Resonance (NMR) Spectroscopy
<ol>
	<li>Prepare 15N-labeled NBB1-156 by standard protein expression and purification as previously described24.</li>
	<li>Dissolve 500 &#181;g of 1,2-distearoyl-sn-glycero-3-phosphoinositol ammonium salt (18:0 PI) and 1,2-dioleoyl-sn-glycero-3-phospho-(1&#39;-myo-inositol) ammonium salt (18:1 PI) in 500 &#181;L of ice-cold&#160;CHCl3 each for final concentrations of 1 mg/mL.</li>
	<li>Sonicate 1 mg/mL 18:0 and 18:1 PI in a sonication water bath for 1&#160;min at room temperature or up to 5 min in an ice-water mixture.</li>
	<li>Aliquot 1 mg/mL 18:0 and 18:1 PI into clean glass vials for individual NMR titration series (Figure 4). Transfer lipids in organic solvents using glass air-tight syringes.
	<ol>
		<li>Aliquot 132 &#181;L of 1 mg/mL 18:1 PI (molar mass = 880.137 g/mol) in CHCl3 for a final resuspension volume of 1,200 &#181;L at 125 &#181;M concentration. 
		NOTE: For 5 mm NMR tubes, prepare serial dilution volumes of &#62;1,000 &#181;L and final NMR sample volumes of &#62;500 &#181;L. For 3 mm NMR tubes, prepare serial dilution volumes of &#62;300 &#181;L and final NMR sample volumes of &#62;150 &#181;L.</li>
	</ol>
	</li>
	<li>Aliquot 1 mg/mL porcine brain L-&#945;-phosphatidylinositol-4,5-bisphosphate ammonium salt in 20:9:1 CHCl3/MeOH/H2O (brain PI(4,5)P2) into clean glass vials.</li>
	<li>Place vials under a gaseous stream of argon (Ar) or nitrogen (N2) with moderate flow to evaporate organic solvent without splashing against the glass vial walls. Five to thirty&#160;min is typically sufficient for volumes of 10&#8211;200 &#181;L organic solvent.</li>
	<li>Arrange glass vial(s) at bottom of a B&#252;chner or vacuum flask using large tweezers, seal with a rubber stopper, and attach plastic tubing connected to a vacuum line. Evacuate flask under mild vacuum overnight to remove residual organics.</li>
	<li>Overlay lipid film aliquots with inert gas, seal with an airtight lid, and store at -20 &#176;C for use within one month or -80 &#176;C for longer-term storage.</li>
	<li>Prepare NMR sample buffers without detergent (-Det) containing 20 mM NaxHyPO4 pH 6.8, 10% D2O and with detergent (+Det) containing 20 mM NaxHyPO4 pH 6.8, 10% D2O, 0.34 mM n-dodecyl-&#946;-D-maltopyranoside (DDM).</li>
	<li>Add +Det buffer to glass vial with lipid film to achieve desired concentration and sonicate in water bath for up to 30 min, alternating sonication for 10 min followed by visual inspection. 
	NOTE: Sonication may warm sample. Allow to cool to room temperature for 15 min before final protein sample reconstitution.</li>
	<li>Perform serial dilution of lipid-detergent micelles in +Det buffer using sonication of 1:1 mixtures for the desired concentration series. Starting at 125 &#181;M lipid concentration, three repetitions will yield 62.5 &#181;M, 31.3 &#181;M, and 15.6 &#181;M lipid in 0.34 mM DDM.</li>
	<li>Prepare control and reference NMR samples of protein and additives in -Det and +Det buffers, respectively. Add protein and additives to each dilution of lipid in +Det buffer. 
	NOTE: For 15N NBB156, protein is added to a final concentration of 40 &#181;M and supplemented with 2 mM deuterated dithiothreitol to keep Cys residues reduced.</li>
	<li>Transfer the appropriate volume of samples to 5 mm or 3 mm NMR tubes (see note in step 5.4).</li>
	<li>Manually load samples in NMR instrument or arrange in an automation platform such as the SampleCase loader.</li>
	<li>Acquire composite NMR spectra using standard pulse programs for 1H proton spectra as quality control followed by 2D 1H-15N correlation spectra either as transverse relaxation optimized spectroscopy (TROSY) or heteronuclear multiple quantum coherence (SOFAST-HMQC)33. 
	NOTE: Usage of 3 mm NMR tubes requires 64 scans for 1H-15N TROSY (~3 h) or 1H-15N SOFAST-HMQC (~90 min). Scan numbers are halved for 5 mm NMR tubes.</li>
	<li>Processed 2D NMR spectra may be added to a CARA repository34 for analysis or other software of choice to the user.</li>
	<li>Analyze software by annotating 2D NMR resonances for three well-dispersed and resolved peaks for each protein state. Record the peak amplitudes for each of the six peaks for each buffer condition containing NBB156 (Figure 5A).
	<ol>
		<li>Use CARA to prepare a batch-integration list (Click Main Menu heading &#34;Spectrum | Setup Batch List&#34;&#8230;) of all spectra collected and analyze&#160;using a single peak assignment file (Click Main Menu heading &#34;Peaks | Import Peaklist&#34;&#160;&#8230;and choose a user-defined .peaks file with 6 total peaks, 3 for each protein state, defined within).</li>
		<li>Tune the settings (&#34;Integrator | Tune Peak Model&#34; &#8230;) of X-width 0.06 ppm and Y-width 0.30 ppm (Click Main Menu heading &#34;Integrator | Tune Peak Model&#34; &#8230;) before recording the final output in two menu selections (1. Click Main Menu heading &#34;Integrator | Integrate Batch List&#34;) (2. Click Main Menu heading &#34;Peaks | Export Integration Table&#34;) (Figure 5B). 
		NOTE: The backbone amide resonances for residues M21, V53, and L105 have been assigned for the NBB156&#160;closed-brace state. The open-brace state was assigned to backbone amide resonances of residues G130 and A141 and the epsilon proton side-chain resonance of W133 using 3D NMR experiments24.</li>
	</ol>
	</li>
	<li>Normalize samples for direct comparison from different magnets or sample conditions by averaging the three open-brace amplitudes of the reference samples and calculating a normalization factor relating the two references. Calculate the scaled amplitudes for NBB156 resonances using the normalization factor and setting negative amplitudes to zero (Table 2). 
	Examples: 1) Comparing samples from different magnets: Magnet A, NBB156+DDM and Magnet B, NBB156+DDM. 2) Detergent comparison: 0.34 mM DDM and 1.7 mM DDM.</li>
	<li>Calculate for each resonance the scaled fraction of closed- or open-brace by dividing with the range of minimum to maximum amplitude of all spectra collected containing appropriate references of free NBB156 and fully open-brace NBB156 in detergent.</li>
	<li>Plot the normalized NMR amplitudes as a function of lipid concentration and compare the fraction of open-brace NBB156 in different conditions (Figure. 5C). 
	NOTE: Alternatively, analysis of the fraction of closed-brace conformation against lipid concentration can be performed to compare lipid binding to NBB156. We prefer the former analysis because it is quicker and better reports on the fraction fully-engaged by lipids.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+3-mediated+necrosis+via+TRIF,+RIP3,+and+MLKL.">Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.</a> Journal of Biological Chemistry. 288 (43), 31268-31279 (2013).</li><li>Upton, J. W., Kaiser, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAI+Another+Way:+Necroptotic+Control+of+Viral+Infection.">DAI Another Way: Necroptotic Control of Viral Infection.</a> Cell Host &amp; Microbe. 21 (3), 290-293 (2017).</li><li>Tanzer, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+signalling+is+tuned+by+phosphorylation+of+MLKL+residues+outside+the+pseudokinase+domain+activation+loop.">Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop.</a> Biochemical Journal. 471 (2), 255-265 (2015).</li><li>Quarato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+Engagement+of+Distinct+MLKL+Phosphatidylinositol-Binding+Sites+Executes+Necroptosis.">Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis.</a> Molecular Cell. 61 (4), 589-601 (2016).</li><li>Davies, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brace+helices+of+MLKL+mediate+interdomain+communication+and+oligomerisation+to+regulate+cell+death+by+necroptosis.">The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis.</a> Cell Death &amp; Differentiation. , (2018).</li><li>Su, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plug+release+mechanism+for+membrane+permeation+by+MLKL.">A plug release mechanism for membrane permeation by MLKL.</a> Structure. 22 (10), 1489-1500 (2014).</li><li>Dondelinger, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MLKL+compromises+plasma+membrane+integrity+by+binding+to+phosphatidylinositol+phosphates.">MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates.</a> Cell Reports. 7 (4), 971-981 (2014).</li><li>Llambi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BOK+Is+a+Non-canonical+BCL-2+Family+Effector+of+Apoptosis+Regulated+by+ER-Associated+Degradation.">BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.</a> Cell. 165 (2), 421-433 (2016).</li><li>Malkani, N., Schmid, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+secrets+of+fluorescent+proteins:+distinct+bleaching+in+various+mounting+fluids+and+photoactivation+of+cyan+fluorescent+proteins+at+YFP-excitation.">Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation.</a> PLoS One. 6 (4), 18586 (2011).</li><li>Perez, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+workflow+for+the+automatic+segmentation+of+organelles+in+electron+microscopy+image+stacks.">A workflow for the automatic segmentation of organelles in electron microscopy image stacks.</a> Frontiers in Neuroanatomy. 8, 126 (2014).</li><li>Walton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lead+aspartate,+an+en+bloc+contrast+stain+particularly+useful+for+ultrastructural+enzymology.">Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology.</a> Journal of Histochemistry &amp; Cytochemistry. 27 (10), 1337-1342 (1979).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLoS Biology. 2 (11), 329 (2004).</li><li>Rossi, P., Xia, Y., Khanra, N., Veglia, G., Kalodimos, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=15N+and+13C-+SOFAST-HMQC+editing+enhances+3D-NOESY+sensitivity+in+highly+deuterated,+selectively+[1H,13C]-labeled+proteins.">15N and 13C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated, selectively [1H,13C]-labeled proteins.</a> Journal of Biomolecular NMR. 66 (4), 259-271 (2016).</li><li>Keller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+computer+aided+resonance+assignment+tutorial.">The computer aided resonance assignment tutorial.</a> Cantina Verlag. , (2004).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+sequence+determinants+control+human+and+mouse+receptor+interacting+protein+3+(RIP3)+and+mixed+lineage+kinase+domain-like+(MLKL)+interaction+in+necroptotic+signaling.">Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling.</a> Journal of Biological Chemistry. 288 (23), 16247-16261 (2013).</li><li>Tanzer, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+of+the+necroptosis+effector+MLKL.">Evolutionary divergence of the necroptosis effector MLKL.</a> Cell Death &amp; Differentiation. 23 (7), 1185-1197 (2016).</li></ol>

We provide protocols for techniques that we combined to implicated MLKL as the putative executioner of plasma membrane rupture24. In addition to deciphering the regulatory network that regulates MLKL-mediated necroptosis, these techniques can be used independently to characterize other suitable biological systems. Practically speaking, these techniques are medium- to low-throughput discovery tools.
We have routinely used live-cell imaging of NBB140-2xFV-Venus...

Identifying genetic components of necroptosis has facilitated the use of animal models to test the implication of necroptosis in physiology and disease1,2,3,4,5. Knockout of RIPK3 or MLKL in mice had minimal implication in development and adult homeostasis suggesting that necroptosis is not essential for life3,6. Moreover, certain species do not contain either RIPK3 or MLKL genes, supporting the non-essential role of necroptosis in animals7,8. On the other hand, challenging knockout animal models with various pathologies induced in the laboratory has revealed an important role of necroptosis in inflammation, innate immunity, and viral infection9,10,11,12.
Necroptosis can be activated in several ways by signaling through different innate immunity sensors, all of which result in the activation of RIPK31,13,14. Active RIPK3 in turn phosphorylates and activates MLKL3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18. The most studied, and perhaps the most complex way, leading to activation of RIPK3 involves death receptor ligation, which bifurcates based on the downstream composition of the signaling complexes to either induce apoptosis or necroptosis1. Necroptosis ensues when signaling through RIPK1 is favored and results in engagement of RIPK319,20. This outcome is easily favored upon pharmacological inhibition or genetic deletion of caspase 8, a putative endogenous inhibitor of necroptosis that keeps necroptosis at bay. RIPK1 binds to and activates RIPK3. Another way to activate necroptosis is through Toll-like receptors TLR3/TLR4 signaling, which engages and activates RIPK3 through TIR-domain-containing adapter-inducing interferon-&#946; (TRIF)21. Yet another way to die by necroptosis is by activation of the DNA sensor DAI, which directly engages and activates RIPK322.
MLKL is a cytosolic protein comprised of an N-terminal helix bundle (NB) domain and a C-terminal pseudokinase domain (psKD) linked by a regulatory brace region3. In normal cells, MLKL is found in the cytosol where it is thought to be in an inactive complex with RIPK314. Activation of necroptosis triggers RIPK3 phosphorylation of MLKL in the activation loop of the psKD, and potentially additional sites in the NB and brace3,15,23. Phosphorylation induces a conformational change in MLKL that results in dissociation from RIPK314. Poorly understood conformational changes release the brace from the psKD24. The brace, which contains 2 helices, mediates oligomerization of MLKL into a putative trimer through the C-terminal helix25. The N-terminal helix of the brace inhibits the NB domain, which is essential for membrane permeabilization24,26. In isolation, NB domain is sufficient to induce plasma membrane permeabilization and necroptosis16,24,27. The pro-necroptotic activity of NB was reconstituted in mouse embryonic fibroblasts deficient in MLKL (mlkl-/- MEFs). NB is a lipid binding domain that preferentially engages the phospholipid phosphatidylinositol 4,5 diphosphate (PIP2). We proposed a stepwise mechanism of activation of MLKL, wherein brace oligomerization facilitates recruitment of MLKL to the plasma membrane via weak interactions of NB with the PIP2 polar head group24. At the membrane, the NB undergoes regulated exposure of an additional high-affinity binding site for PIP2, which is masked by the brace in inactive MLKL. Overall, the multiple interactions of NB with PIP2 destabilize the plasma membrane leading to its rupture, although the molecular mechanism of these events have not been elucidated.
Here we illustrate specific methods used to characterize the function of MLKL as executioner of necroptosis24. In particular, we focus on the most minimal domain of MLKL, the NB and brace (NBB), which is regulated by brace inhibition and can be activated through enforced dimerization to induce plasma membrane rupture and necroptosis. We describe our inducible expression system combined with enforced drug-induced FKBP-mediated dimerization for live-cell imaging, and electron microscopy of cells undergoing necroptosis. Additionally, we illustrate our in vitro NMR analysis of the interactions of NBB with phosphatidylinositols (PIPs).

<table><tbody><tr><td>&lt;strong&gt;Cloning and cell line generation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pRetroX-TRE3G</td><td>Clontech</td><td>631188</td><td></td></tr><tr><td>Tet-On transactivator plasmid</td><td></td><td></td><td>Llambi &lt;em&gt;et al.&lt;/em&gt;, 2016</td></tr><tr><td>Mouse Embryonic Fibroblasts (MEFs) mlkl-/-</td><td></td><td></td><td>Dillon &lt;em&gt;et al.&lt;/em&gt;, 2014</td></tr><tr><td>Blasticidin S Hydrochloride</td><td>Thermo Fisher Scientific</td><td>BP2647100 CAS#3513-03-9</td><td></td></tr><tr><td>&lt;strong&gt;Cell death quantification and live-cell microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Doxycycline</td><td>Clontech</td><td>631311 CAS# 24390-14-5</td><td></td></tr><tr><td>B/B Homodimerizer AP20187</td><td>Takara</td><td>635059 CAS# 195514-80-8</td><td></td></tr><tr><td>SYTOX Green</td><td>Thermo Fisher Scientific</td><td>S7020</td><td></td></tr><tr><td>Syto16</td><td>Thermo Fisher Scientific</td><td>S7578</td><td></td></tr><tr><td>&lt;strong&gt;NMR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 N Ammonium Chloride</td><td>Cambridge Isotope Laboratories</td><td>NLM-467-10 CAS# 12125-02-9</td><td></td></tr><tr><td>Deuterated DTT</td><td>Cambridge Isotope Laboratories</td><td>DLM-2622-1</td><td></td></tr><tr><td>Deuterium Oxide</td><td>Sigma Aldrich</td><td>617385-1 CAS# 7789-20-0</td><td></td></tr><tr><td>n-Dodecyl-&amp;beta;-D-Maltopyranoside</td><td>Anatrace</td><td>D310 CAS# 69227-93-6</td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt)</td><td>Avanti Polar Lipids</td><td>840046X CAS# 383907-42-4</td><td></td></tr><tr><td>1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (18:0 PI)</td><td>Avanti Polar Lipids</td><td>850143 CAS# 849412-67-5</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phospho-(1&#039;-myo-inositol) (ammonium salt) (18:1)</td><td>Avanti Polar Lipids</td><td>850149 CAS# 799268-53-4</td><td></td></tr><tr><td>&lt;strong&gt;Specialized Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IncuCyte FLR or ZOOM</td><td>Essen BioScience, Inc.</td><td></td><td>Live-cell microscopy imaging</td></tr><tr><td>Helios NanoLab 660 DualBeam&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td></td><td>Electron microscope</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IncuCyte 2011A Rev2 v20111.3.4288 (FLR)</td><td>Essen BioScience, Inc.</td><td>http://www.essenbioscience.com</td><td>Imaging analysis</td></tr><tr><td>FEI MAPS</td><td>Thermo Fisher Scientific</td><td>https://www.fei.com/software/maps/</td><td>EM analysis</td></tr><tr><td>TopSpin v3.2</td><td>Bruker BioSpin</td><td>http://www.bruker.com</td><td>NMR data collection</td></tr><tr><td>CARA v1.9.1.7</td><td></td><td>http://cara.nmr.ch/&amp;nbsp;</td><td>NMR data analysis</td></tr><tr><td>Slidebook</td><td>3i (Intelligent Imaging Innovations)</td><td>https://www.intelligent-imaging.com/slidebook</td><td>Confocal microscopy</td></tr></tbody></table>

characterization of mlkl-mediated plasma membrane rupture in necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

Visualizing regulated necroptosis execution in live cells has been possible through inducible expression of a minimal truncated MLKL construct, NBB140-2xFV-Venus. This construct maintains the ability to induce plasma membrane permeabilization and is activated through Dim-induced oligomerization of the FKBP cassette (2xFV). We observe and quantify necroptosis by live-cell microscopy imaging, monitoring kinetically (every 5 min) the uptake of a cell impermeable green fluorescence...

Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

characterization-mlkl-mediated-plasma-membrane-rupture

Research

JoVE Journal

Biology

10.8K Views.  St. Jude Children's Research Hospital. Wij rapporteren methoden voor karakterisering van MLKL-gemedieerde plasmamembraan breuk in necroptosis met inbegrip van conventionele en confocal microscopie van live-cel imaging, scanning elektronen microscopie, en NMR gebaseerde lipide bindend.

Video: Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Cancer Research

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

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Neuroscience

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical ...

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 ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

Analyzing LPS/ATP-Triggered Cell Death Mechanisms in THP-1 Cells

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show&#160;that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

Author Spotlight: THP-1 Macrophage Response to LPS/ATP &#8212; Unveiling the Pyroptosis, Apoptosis, and Necroptosis Spectrum

This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

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