Name: characterization of mlkl-mediated plasma membrane rupture in necroptosis
Uploaded: 2018-08-07T15:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

<ol>
	<li>Weinlich, R., Oberst, A., Beere, H. M., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+in+development,+inflammation+and+disease.">Necroptosis in development, inflammation and disease.</a> Nature Reviews Molecular Cell Biology. 18 (2), 127-136 (2017).</li><li>Kaiser, W. 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=RIP3+mediates+the+embryonic+lethality+of+caspase-8-deficient+mice.">RIP3 mediates the embryonic lethality of caspase-8-deficient mice.</a> Nature. 471 (7338), 368-372 (2011).</li><li>Murphy, J. M., 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+pseudokinase+MLKL+mediates+necroptosis+via+a+molecular+switch+mechanism.">The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.</a> Immunity. 39 (3), 443-453 (2013).</li><li>Oberst, 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=Catalytic+activity+of+the+caspase-8-FLIP(L)+complex+inhibits+RIPK3-dependent+necrosis.">Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.</a> Nature. 471 (7338), 363-367 (2011).</li><li>Zhang, H., 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=Functional+complementation+between+FADD+and+RIP1+in+embryos+and+lymphocytes.">Functional complementation between FADD and RIP1 in embryos and lymphocytes.</a> Nature. 471 (7338), 373-376 (2011).</li><li>He, S., 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=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137 (6), 1100-1111 (2009).</li><li>Dondelinger, Y., Hulpiau, P., Saeys, Y., Bertrand, M. J. M., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolutionary+perspective+on+the+necroptotic+pathway.">An evolutionary perspective on the necroptotic pathway.</a> Trends in Cell Biology. 26 (10), 721-732 (2016).</li><li>Newton, K., Manning, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+and+Inflammation.">Necroptosis and Inflammation.</a> Annual Review of Biochemistry. 85, 743-763 (2016).</li><li>Kaiser, W. J., Upton, J. W., Mocarski, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+modulation+of+programmed+necrosis.">Viral modulation of programmed necrosis.</a> Current Opinion in Virology. 3 (3), 296-306 (2013).</li><li>Newton, K., 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=RIPK3+deficiency+or+catalytically+inactive+RIPK1+provides+greater+benefit+than+MLKL+deficiency+in+mouse+models+of+inflammation+and+tissue+injury.">RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.</a> Cell Death &amp; Differentiation. 23 (9), 1565-1576 (2016).</li><li>Kearney, C. J., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Inflammatory+Perspective+on+Necroptosis.">An Inflammatory Perspective on Necroptosis.</a> Molecular Cell. 65 (6), 965-973 (2017).</li><li>Pasparakis, M., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+and+its+role+in+inflammation.">Necroptosis and its role in inflammation.</a> Nature. 517 (7534), 311-320 (2015).</li><li>Sun, L., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+kind+of+cell+suicide:+mechanisms+and+functions+of+programmed+necrosis.">A new kind of cell suicide: mechanisms and functions of programmed necrosis.</a> Trends in Biochemical Sciences. 39 (12), 587-593 (2014).</li><li>Grootjans, S., Vanden Berghe, T., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+and+execution+mechanisms+of+necroptosis:+an+overview.">Initiation and execution mechanisms of necroptosis: an overview.</a> Cell Death &amp; Differentiation. 24 (7), 1184-1195 (2017).</li><li>Sun, 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=Mixed+lineage+kinase+domain-like+protein+mediates+necrosis+signaling+downstream+of+RIP3+kinase.">Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.</a> Cell. 148 (1-2), 213-227 (2012).</li><li>Wang, H., 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=Mixed+lineage+kinase+domain-like+protein+MLKL+causes+necrotic+membrane+disruption+upon+phosphorylation+by+RIP3.">Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.</a> Molecular Cell. 54 (1), 133-146 (2014).</li><li>Cai, Z., 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=Plasma+membrane+translocation+of+trimerized+MLKL+protein+is+required+for+TNF-induced+necroptosis.">Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.</a> Nature Cell Biology. 16 (1), 55-65 (2014).</li><li>Chen, X., 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=Translocation+of+mixed+lineage+kinase+domain-like+protein+to+plasma+membrane+leads+to+necrotic+cell+death.">Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death.</a> Cell Research. 24 (1), 105-121 (2014).</li><li>Dillon, C. P., 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=RIPK1+blocks+early+postnatal+lethality+mediated+by+caspase-8+and+RIPK3.">RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.</a> Cell. 157 (5), 1189-1202 (2014).</li><li>Rickard, J. 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=RIPK1+regulates+RIPK3-MLKL-driven+systemic+inflammation+and+emergency+hematopoiesis.">RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.</a> Cell. 157 (5), 1175-1188 (2014).</li><li>Kaiser, W. 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>

膜破断24の推定死刑執行人として MLKL に関与する、我々 を組み合わせた技術のためのプロトコルを提供します。MLKL を介したネクロトーシスを調節する制御ネットワークを解読する、に加えてこれらのテクニックがない独立して使用できる他適切な生物学的システムを特徴付けるため。実質的に言えば、これらのテクニックは、中-低スループット探索ツールです。
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ネクロトーシスの遺伝的要素を識別する生理・疾患1,2,3,4,の5ネクロトーシスの含意をテストするための動物モデルの使用を促進しています。RIPK3 または MLKL のノックアウト マウスは、そのネクロトーシスは人生3,6の必須ではないことを示唆開発および大人の恒常性で最小限の含意を持っていた。さらに、特定の種は動物7,8ネクロトーシスの非本質的な役割をサポート、RIPK3 または MLKL のいずれかの遺伝子を含まない。その一方で、研究室に誘導される様々 な病態とノックアウト動物モデルに挑戦、炎症、免疫、ウイルス感染9,10,ネクロトーシスの重要な役割が明らかに11,12。
ネクロトーシスは異なる免疫センサー信号によっていくつかの方法で実行されるすべての RIPK31,13,14の活性化に繋がる。アクティブな RIPK3 化順番 MLKL3,4,5,6,7,8,9,10 をアクティブにし、、11,12,13,14,15,16,17,18。ほとんどが勉強した、おそらく最も複雑な方法は、RIPK3 の活性化につながる死受容体血管を結紮を分割するか apoptosis かネクロトーシス1を誘導するシグナル伝達複合体の下流の組成に基づくです。ネクロトーシスはすさまじいが好まれている RIPK1 を介したシグナル伝達機構と RIPK319,20の関与の結果です。この結果は、カスパーゼ 8、ベイでネクロトーシスを保持ネクロトーシスの推定される内因性阻害剤の薬理学的抑制遺伝的削除に簡単に優先されます。RIPK1 に結合し、RIPK3 がアクティブになります。ネクロトーシスをアクティブにする別の方法は、従事し、TIR ドメインを含むアダプター誘導インターフェロン-β (TRIF)21を介して RIPK3 をアクティブにする Toll 様受容体 TLR3/TLR4 シグナル伝達を介してです。まだネクロトーシスで死ぬことの別の方法は、直接従事し、RIPK322をアクティブに DNA センサー台の活性化です。
MLKL N 末端ヘリックス バンドル (NB) ドメインと規制ブレース地域3によってリンクされている C 末端 pseudokinase ドメイン (psKD) から成る細胞内蛋白質であります。正常細胞の MLKL はどこ RIPK314と非アクティブな複合体であると考えられる細胞質にあります。ネクロトーシスの活性化は、psKD、および NB とブレースの3,15,23で可能性のある追加のサイトの活発化のループの MLKL の RIPK3 のリン酸化をトリガーします。リン酸化は、RIPK314から解離による MLKL の構造変化を誘導します。不十分な理解の構造変化は、psKD24からブレースを解放します。2 本のヘリックスが含まれ、ブレースは、C ターミナル螺旋形25推定三量体に MLKL の重合を仲介します。ブレースの N 末端ヘリックスは、膜透過24,26のために不可欠である NB ドメインを阻害します。分離、NB ドメインは細胞膜透過とネクロトーシス16,24,27を誘導するために十分です。NB の pro necroptotic 活性は、マウス胚性線維芽細胞 (mlkl-/- MEFs) MLKL 欠損で再構成されました。NB は優先的に従事するリン脂質ホスファチジルイノシトール 4, 5 二リン酸 (PIP2) 脂質結合ドメインです。ブレース重合が PIP2極性基24NB の弱い相互作用を介して細胞膜と MLKL の採用を促進する前記 MLKL の活性化の段階的な機構を提案する.膜では、NB は、PIP2のため非アクティブな MLKL 内のブレースによってマスクされます追加の高親和性結合サイトの規制の露出を経る。全体的にみて、PIP2と NB の複数の相互作用は、これらのイベントの分子メカニズムが解明されていないが膜の破断につながる不安定します。
ここで我々 はネクロトーシス24の死刑執行人として MLKL の機能を特徴付けるために使用する特定のメソッドを示しています。特に、我々 は MLKL, NB, ブレース (NBB) ブレース阻害によって規制されており膜破裂とネクロトーシスを誘発する強制二量体化してアクティブになることができますの最も最小限のドメインに焦点を当てます。強制薬物誘発性 FKBP を介した二量体化住セルイメージ投射とネクロトーシスになる細胞の電子顕微鏡観察のために結合した発現システムについて述べる。さらに、NBB フォスファチジルイノシトール (PIPs) との相互作用の in vitro NMR 分析を示す.

<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

ネクロトーシスはし、混合系統キナーゼのようなドメインの pseudokinase、MLKL の破裂や permeabilize 血しょう膜を活性化するタンパク質キナーゼ 3 (RIPK3) を相互作用する受容体の活性化によって引き起こされるプログラムされた細胞死経路.ネクロトーシスは自己免疫、感染症や心血管系疾患や脳卒中、神経変性、がんを含む複数の疾患に関連付けられている炎症経路です。ここでは、ネクロトーシス膜破裂の死刑執行人として MLKL を特徴付けるために使用できるプロトコルについて述べる。我々 はネクロトーシス従来および共焦点蛍光顕微鏡を用いた生細胞イメージングを用いた細胞およびプラズマへの細胞質から MLKL の再分配を一緒に明らかに電子顕微鏡を用いた固定細胞プロセスを視覚化します。血しょう膜の大きな穴の誘導の前に膜。MLKL を介したネクロトーシスの推定される変調器を識別するために脂質を用いた核磁気共鳴 (NMR) 分析体外を提案します。本法に基づき、特定定量的脂質結合の好みホスファチジルコリン イノシトールリン酸 (PIPs) 膜ターゲットとネクロトーシスに透過に必要な MLKL の重要なバインダーとしてします。

最小限切り捨てられた MLKL 構築、NBB140の発現誘導を介して可能にされている生きているセルの規制ネクロトーシス実行を可視化する-2xFV-金星。このコンス トラクターは、膜透過を誘導する能力を維持し、FKBP カセット (2xFV) Dim 誘起重合を介して活性化します。我々 を観察して生細胞顕微鏡イメージング、監視速度論的 (5 分ごと) 細胞不浸透性の緑色蛍光 DNA ?...

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

走査型電子顕微鏡および NMR による脂質結合ネクロトーシス従来および共焦点の生細胞顕微鏡イメージングなどの MLKL を介した膜破断の法を述べる。

ネクロトーシス MLKL を介した細胞膜破壊の解析

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 ビュー.  St. Jude Children's Research Hospital. 走査型電子顕微鏡および NMR による脂質結合ネクロトーシス従来および共焦点の生細胞顕微鏡イメージングなどの MLKL を介した膜破断の法を述べる。

ビデオ: 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.

CLIC4 ノックダウン誘起 HN4 細胞アポトーシスの in Vitro研究にミトコンドリア膜電位の検出

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

がん研究

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.

インフラマソーム活性化とマウス骨髄由来マクロファージにおける Pyroptotic の細胞死の検出

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

免疫・感染学

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.

神経細胞死とマウス プライマリ小脳顆粒ニューロンの変性をモデリング

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

神経科学

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.

哺乳類細胞における効率を再封止膜の高スループット測定

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.

ヒト細胞におけるHSV-1感染後のRIPK3およびMLKLのZBP1依存性リン酸化の免疫蛍光染色のためのチラミドシグナル増幅

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.

自然免疫細胞死を評価するためのカスパーゼ活性化の評価

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.

隣接セル受けてアポトーシス細胞死との相互作用によって生細胞に誘導されるイベントを細胞内シグナルの同定

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

生物学

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.

CLON-Gおよび in vitro 自然死アッセイによる好中球の寿命延長

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 ...

THP-1細胞におけるLPS/ATP誘発細胞死メカニズムの解析

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.

著者スポットライト:LPS/ATPに対するTHP-1マクロファージ応答 — ピロトーシス、アポトーシス、ネクロプトーシススペクトルの解明

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.

PMA分化型THP-1マクロファージのLPSおよびATP誘発死とその検証

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

ネクロトーシス MLKL を介した細胞膜破壊の解析

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この動画の章

CLIC4 ノックダウン誘起 HN4 細胞アポトーシスの in Vitro研究にミトコンドリア膜電位の検出

インフラマソーム活性化とマウス骨髄由来マクロファージにおける Pyroptotic の細胞死の検出

神経細胞死とマウスプライマリ小脳顆粒ニューロンの変性をモデリング

哺乳類細胞における効率を再封止膜の高スループット測定

ヒト細胞におけるHSV-1感染後のRIPK3およびMLKLのZBP1依存性リン酸化の免疫蛍光染色のためのチラミドシグナル増幅

自然免疫細胞死を評価するためのカスパーゼ活性化の評価

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

隣接セル受けてアポトーシス細胞死との相互作用によって生細胞に誘導されるイベントを細胞内シグナルの同定

CLON-Gおよび in vitro 自然死アッセイによる好中球の寿命延長

PMA分化型THP-1マクロファージのLPSおよびATP誘発死とその検証

ネクロトーシス MLKL を介した細胞膜破壊の解析

要約

さらに動画を探す

この動画の章

CLIC4 ノックダウン誘起 HN4 細胞アポトーシスの in Vitro研究にミトコンドリア膜電位の検出

インフラマソーム活性化とマウス骨髄由来マクロファージにおける Pyroptotic の細胞死の検出

神経細胞死とマウス プライマリ小脳顆粒ニューロンの変性をモデリング

哺乳類細胞における効率を再封止膜の高スループット測定

ヒト細胞におけるHSV-1感染後のRIPK3およびMLKLのZBP1依存性リン酸化の免疫蛍光染色のためのチラミドシグナル増幅

自然免疫細胞死を評価するためのカスパーゼ活性化の評価

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

隣接セル受けてアポトーシス細胞死との相互作用によって生細胞に誘導されるイベントを細胞内シグナルの同定

CLON-Gおよび in vitro 自然死アッセイによる好中球の寿命延長

PMA分化型THP-1マクロファージのLPSおよびATP誘発死とその検証

神経細胞死とマウスプライマリ小脳顆粒ニューロンの変性をモデリング