This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

発現プラスミドpHLsec-Gzm 1.生産<ol><li>適切なRNA単離法を用いて、ヒトNK細胞（一次細胞は、すべての5人のGzmsを発現するNK-92 26またはNK細胞株のように調製した）からの総RNAを調製し、第一鎖cDNAを合成しキットを用いての逆転写反応、manufacturer`以下の提言。 23（ラドゥAricescuとイヴォンヌ·ジョーンズの種類のギフト、オックスフォード、英国の大学）に記載されているように（ 図1）をAgeI及びKpnI部位を使用してpHLsecにPCRおよびクローンを使用してGzm cDNAを増幅。 注： 表1に示し、以下のプライマーを使用してください。 </li><li>配列決定によって正しい挿入を確認してください。 DH5α細胞中で発現プラスミドを展開し、エンドトキシンフリープラスミド単離キットを用いて精製し、製造者の指示に従ってください。 </li><li>を2mg / mlの濃度でエンドトキシンフリー滅菌水で精製したプラスミドを再懸濁し、店で-20°C untiL使用。 </li></ol> HEK293T細胞におけるGzmsの2式<ol><li>試薬の調製<ol><li>標準的な培養培地を準備します。ダルベッコ改変イーグル培地（DMEM）を含有する高グルコース（25ミリモル）、グルタマックス（4ミリモル）に、ピルビン酸ナトリウム（1mM）を（標準のウシ胎児血清（FCS）、ペニシリン（100単位/ ml）、ストレプトマイシンの10％を加えます100μg/ mlの）。 </li><li>トランスフェクション培地を準備します。ペニシリン - ストレプトマイシンを含まない培地に25 / mlのクロロキン（-20℃で保存し、PBSで1,000倍の株式、からトランスフェクションの日に、新たに追加）を追加します。 </li><li>無血清培地を準備します。無血清培地にHEK293細胞は、グルタミン（4 mM）の、ペニシリン（100単位/ ml）、ストレプトマイシン（100μg/ mlの）、および2.5mgの/ LのアンホテリシンBを追加するため</li><li> 表2に記載されたソリューションを用意し、使用前に0.45μmのフィルターでフィルター： </li></ol></li><li> HEK293T細胞を拡大<ol><li> 20ミリリットルカルトにHEK293T細胞を成長させます150×25 mm組織培養皿を用いてURE媒体。 </li><li> 80％の密集度で分割細胞（1のスプリット比：4、通常3日毎）。細胞が緩く取り付け、それらは機械的に厳密に上下にピペッティングによりトリプシン処理せずに取り外すことができます。 </li><li>プレートの細胞は、トランスフェクション前日に、トランスフェクション（播種密度約2×10 5細胞/ ml）の日に60〜70％の集密度を得ました。通常の準備のサイズは20〜25培養皿で構成されています。 </li></ol></li><li>リン酸カルシウムトランスフェクション<ol><li>トランスフェクションの1時間前には、20ミリリットルのトランスフェクション培地に標準的な培養培地を変更します。この1時間のインキュベーション工程の終わり近く、10.95ミリリットルののddH 2 0〜50 mlチューブ中2MのCaCl 2の1.55ミリリットル（1チューブ/ 5皿）でプラスミドDNA400μgのを混ぜます。 </li><li>トランスフェクションの前に1〜2分、DNA-カルシウム溶液12.5 mlに2×HBS 12.5ミリリットルを加え、チューブの反転により混合し、室温で30秒間インキュベートします。 </li><li>目を追加します。Eの混合物を滴下培地中に、細胞（皿当たり5ミリリットル）に直接2.3.2に用意しました。ややオレンジ色に培地を回し、全領域に均等に振りかけます。 </li><li>組織培養インキュベーター（37℃、加湿空気中5％CO 2）で7から11時間、培養皿をインキュベートします。インキュベーション後、微細な沈殿物が見えます。予め温めておいたPBSで丁寧に洗い流し、トランスフェクション培地を除去し、2.1.3で調製した20ミリリットルの無血清培地を追加します。組織培養インキュベーター中で72時間インキュベートします。 </li><li> SDS-PAGE上での細胞の上清のサンプル（〜20μl）を実行し、グランザイムバンドを可視化するために、クマシーブリリアントブルーで染色することにより、トランスフェクション及び発現効率を分析します。 </li></ol></li><li>ニッケルIMACにより培養上清からのGzmsの精製<ol><li>インキュベーション後、250ミリリットルチューブに細胞培養上清をデカントし、遠心分離により明確。最初のスピンを行う（400×gで、10分間、4°剥離した細胞からの培地をクリアしますC）。残りの細胞破片を除去し、4℃で4,000×gで、30分で新鮮な250ミリリットルチューブとスピンで上清を転送します。 </li><li> 5 M NaClを5mlの、透明な上清の250ミリリットル当たり0.25 MのNiSO 4の6.25 2 M Tris塩基（pHは8）のミリリットルと1ミリリットルを追加します。 500ミリリットル真空フィルターユニット（0.45 mm）を用いて上清をフィルタリングします。 </li><li>適切なFPLCシステムを使用して彼の結合緩衝液A（10カラム体積、CV）とニッケルIMACカラムを平衡化します。 </li><li> 4℃で0.5ml /分の流速で平衡化したカラムに透明な上清を適用します。 </li><li>上清を適用した後、UV吸光度がベースラインに達するまで、（通常、10 CV）のHis結合バッファーでカラムを洗浄します。 </li><li> 0.5ml /分の流速で20ミリリットルの線形勾配（0~250 mMイミダゾール）で溶出Gzms 2ミリリットル画分を収集しました。 SDS-PAGEおよびクーマシー染色により溶出画分を分析します。 </li></ol></li><li>エンテロキナーゼ（EK）治療および陽イオン交換クロマトグラフィーによる最終クリーンアップ<ol><li> ≤10MWCOに透析チューブ内の画分を含むプールGzm。前EK制御などの-20℃での少量のサンプルを保管してください。 </li><li>透析チューブに直接にプール画分に0.02単位/初期上清を50 mlのEKを追加して、O / N EK-バッファーで室温、（少なくとも16時間）（4 L、一度変更バッファ）を透析。 </li><li>次の日、EKは、SDS-PAGEおよびクマシー染色による事前EKコントロールと比較してGzmsを処理し分析します。 </li><li> N末端の処理が完了すると、SバッファーA（4 L）に透析緩衝液を変更して、室温でさらに4時間透析します。フィルター透析液（0.45ミクロン）。 </li><li> 4℃で0.5ml /分の流速でカラムにサンプルをロードS緩衝液AでS-カラムを平衡化します。 </li><li> UV吸光度のベースラインに達するまで、試料ローディングの後、（約10 CV）S緩衝液Aでカラムを洗浄します。 30ミリリットルの直線勾配（150〜1000 mMのNaCl）でGzmsを溶出。 GZMA E〜750mMのNaClで〜700mMのNaClおよびGzmMで〜650のNaCl、GZMBでリュート。 </li><li> SDS-PAGEおよび比色アッセイ（下記参照）による溶出画分を分析します。 Gzmsおよび濃縮物を含むプール画分（約30倍、約100μMの濃度に）スピンフィルターで（15ミリリットル、10 MWCO）。 -80℃で濃縮さGzm製剤およびストアを等分。 </li></ol></li></ol> 3.テストGzm活動<ol><li>試薬の調製<ol><li> 50mMのトリス塩基、pHは7：比色アッセイ緩​​衝液を準備します。 <ol><li> GZMAの測定のために、0.2mMの及び5,5&#39;-ジチオ - ビス（2-ニトロ安息香酸）の最終濃度までアッセイ緩​​衝液へのNa-CBZ-L-リジンをチオベンジルエステル（S-Bzlの）（BLT）を追加0.22 mMの濃度に（DTNB）。 GZMBについては、0.2 mMののBoc-ALA-ALA-ASP-S-Bzlの（AAD）および0.22 mMのDTNBを含みます。 GzmMについては、0.2 mMのあるSuc-ALA-ALA-PRO-Leuの-p-ニトロアニリド（PNA）（AAPL）が挙げられます。合成ペプチドは、DMSO中で（適切なストック溶液から追加され、STORアッセイの前に、新たにバッファに）-20°Cで編。 </li></ol></li><li>切断アッセイバッファーを準備します。100mMのNaCl、50mMのトリス塩基、pHは7.5 </li></ol></li><li>比色アッセイ<ol><li> 96ウェル平底プレート中の溶出画分または濃縮されたストックから小さなGzmサンプル（&lt;5μl）を配布します。ポジティブコントロール（試験したGzm製剤または粗製のNK細胞溶解物）および陰性コントロール（緩衝液のみ）も含めます。 </li><li> （マルチチャンネルピペットを使用して）各ウエルに、アッセイ緩​​衝液100μlを加えます。 37℃で10分間インキュベートします。マイクロプレートリーダーで405nmでODを測定します。 </li></ol></li><li>切断アッセイ<ol><li>精製された基板を使用して切断アッセイのために、タンパク質基質を共インキュベート（天然または組換え、100から400 nMの、効率的なGzm基板のいくつかの例は、このセクションの最後に結果の項でノートに示されている）の連続希釈でGzmの様々な時間のためにアッセイ緩​​衝液で（400nmで開始します）。SDS-PAGEおよびクーマシーまたは銀染色によって分析します。または特異的抗体を用いたウェスタンブロッティングによって。 </li><li>細胞溶解物を使用して切断アッセイのために、37℃で三回でエタノール/ドライアイス浴融解に凍結することによってアッセイ緩衝液と溶解1ml中10 6のHeLa細胞（または任意の利用可能な腫瘍細胞株）をサスペンド。 （4°Cで10分間15,000×gで）を、遠心分離によって細胞破片を除去。 </li><li>上記のように様々な濃度と時間でGzmsでクリアした溶解物を共インキュベートします。切断は、適切な抗体を用いたイムノブロッティングによって検出されます。 注：市販の抗体が利用可能な善意の基板のいくつかの例：GZMB 27,28によって切断入札（BH3相互作用ドメイン死アゴニスト）およびカスパーゼ3; GZMA 29によって切断され、複数の異種核リボ（hnRNPA1、A2およびU）; GzmM 30によるサイトメガロウイルスリンタンパク質71。 </li></ol></li></ol>

<ol>
	<li>Chowdhury, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+by+a+thousand+cuts:+granzyme+pathways+of+programmed+cell+death.">Death by a thousand cuts: granzyme pathways of programmed cell death.</a> Annu Rev Immunol. 26, 389-420 (2008).</li><li>Thiery, J., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin:+a+key+pore-forming+protein+for+immune+control+of+viruses+and+cancer.">Perforin: a key pore-forming protein for immune control of viruses and cancer.</a> Subcell Biochem. 80, 197-220 (2014).</li><li>Krensky, A. M., Clayberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+clinical+relevance+of+granulysin.">Biology and clinical relevance of granulysin.</a> Tissue Antigens. 73, 193-198 (2009).</li><li>Bovenschen, N., Kummer, 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=Orphan+granzymes+find+a+home.">Orphan granzymes find a home.</a> Immunol Rev. 235, 117-127 (2010).</li><li>Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+A+activates+another+way+to+die.">Granzyme A activates another way to die.</a> Immunol Rev. 235, 93-104 (2010).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quarter+century+of+granzymes.">A quarter century of granzymes.</a> Cell death and differentiation. 19, 28-35 (2012).</li><li>Hiebert, P. R., Granville, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B+in+injury,+inflammation,+and+repair.">Granzyme B in injury, inflammation, and repair.</a> Trends Mol Med. 18, 732-741 (2012).</li><li>Afonina, I. S., Cullen, S. P., 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=Cytotoxic+and+non-cytotoxic+roles+of+the+CTL/NK+protease+granzyme.">Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme.</a> B. Immunol Rev. 235, 105-116 (2010).</li><li>Walch, 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=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157, 1309-1323 (2014).</li><li>Lee, W. 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=Invariant+natural+killer+T+cells+act+as+an+extravascular+cytotoxic+barrier+for+joint-invading+Lyme+Borrelia.">Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia.</a> Proc Natl Acad Sci U S A. , (2014).</li><li>Kapelski, S., de Almeida, M., Fischer, R., Barth, S., Fendel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimalarial+activity+of+granzyme+B+and+its+targeted+delivery+by+a+granzyme+B-scFv+fusion+protein.">Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-scFv fusion protein.</a> Antimicrob Agents Chemother. , (2014).</li><li>Thiery, J., Walch, M., Jensen, D. K., Martinvalet, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+cytotoxic+T+cell+and+NK+granules+and+purification+of+their+effector+proteins.">Isolation of cytotoxic T cell and NK granules and purification of their effector proteins.</a> Curr Protoc Cell Biol. Chapter 3 (Unit3 37), (2010).</li><li>Shi, L., Yang, X., Froelich, C. J., Greenberg, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+use+of+granzyme+B.">Purification and use of granzyme B.</a> Methods Enzymol. 322, 125-143 (2000).</li><li>Masson, D., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+serine+esterases+in+lytic+granules+of+cytolytic+T+lymphocytes.">A family of serine esterases in lytic granules of cytolytic T lymphocytes.</a> Cell. 49, 679-685 (1987).</li><li>Lorentsen, R. H., Fynbo, C. H., Thogersen, H. C., Etzerodt, M., Holtet, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+refolding,+and+purification+of+recombinant+human+granzyme+B.">Expression, refolding, and purification of recombinant human granzyme B.</a> Protein Expr Purif. 39, 18-26 (2005).</li><li>Sun, 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=Expression+and+purification+of+recombinant+human+granzyme+B+from+Pichia+pastoris.">Expression and purification of recombinant human granzyme B from Pichia pastoris.</a> Biochem Biophys Res Commun. 261, 251-255 (1999).</li><li>Xia, 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=Expression+and+purification+of+enzymatically+active+recombinant+granzyme+B+in+a+baculovirus+system.">Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.</a> Biochem Biophys Res Commun. 243, 384-389 (1998).</li><li>Stahnke, B., 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=Granzyme+B-H22(scFv),+a+human+immunotoxin+targeting+CD64+in+acute+myeloid+leukemia+of+monocytic+subtypes.">Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes.</a> Mol Cancer Ther. 7, 2924-2932 (2008).</li><li>Gehrmann, 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=A+novel+expression+and+purification+system+for+the+production+of+enzymatic+and+biologically+active+human+granzyme.">A novel expression and purification system for the production of enzymatic and biologically active human granzyme.</a> B. J Immunol Methods. 371, 8-17 (2011).</li><li>Metkar, S. 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=Cytotoxic+cell+granule-mediated+apoptosis:+perforin+delivers+granzyme+B-serglycin+complexes+into+target+cells+without+plasma+membrane+pore+formation.">Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation.</a> Immunity. 16, 417-428 (2002).</li><li>Motyka, B., 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=Mannose+6-phosphate/insulin-like+growth+factor+II+receptor+is+a+death+receptor+for+granzyme+B+during+cytotoxic+T+cell-induced+apoptosis.">Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis.</a> Cell. 103, 491-500 (2000).</li><li>Pinkoski, M. 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=Entry+and+trafficking+of+granzyme+B+in+target+cells+during+granzyme+B-perforin-mediated+apoptosis.">Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.</a> Blood. 92, 1044-1054 (1998).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).</li><li>Fukuchi, 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=Activity+assays+of+nine+heterogeneous+promoters+in+neural+and+other+cultured+cells.">Activity assays of nine heterogeneous promoters in neural and other cultured cells.</a> In Vitro Cell Dev Biol Anim. 30 A, 300-305 (1994).</li><li>Tran, T. V., Ellis, K. A., Kam, C. M., Hudig, D., Powers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dipeptidyl+peptidase+I:+importance+of+progranzyme+activation+sequences,+other+dipeptide+sequences,+and+the+N-terminal+amino+group+of+synthetic+substrates+for+enzyme+activity.">Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity.</a> Arch Biochem Biophys. 403, 160-170 (2002).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Barry, 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=Granzyme+B+short-circuits+the+need+for+caspase+8+activity+during+granule-mediated+cytotoxic+T-lymphocyte+killing+by+directly+cleaving+Bid.">Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid.</a> Mol Cell Biol. 20, 3781-3794 (2000).</li><li>Darmon, A. J., Nicholson, D. W., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+apoptotic+protease+CPP32+by+cytotoxic+T-cell-derived+granzyme+B.+.">Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. .</a> Nature. 377, 446-448 (1995).</li><li>Rajani, D. K., Walch, M., Martinvalet, D., Thomas, M. P., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+RNA+processing+during+immune-mediated+programmed+cell+death.">Alterations in RNA processing during immune-mediated programmed cell death.</a> Proc Natl Acad Sci U S A. 109, 8688-8693 (2012).</li><li>Domselaar, R., 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=Noncytotoxic+inhibition+of+cytomegalovirus+replication+through+NK+cell+protease+granzyme+M-mediated+cleavage+of+viral+phosphoprotein+71.">Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.</a> J Immunol. 185, 7605-7613 (2010).</li><li>Thiery, 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=Perforin+activates+clathrin-+and+dynamin-dependent+endocytosis,+which+is+required+for+plasma+membrane+repair+and+delivery+of+granzyme+B+for+granzyme-mediated+apoptosis.">Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.</a> Blood. 115, 1582-1593 (2010).</li><li>Thiery, 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=Perforin+pores+in+the+endosomal+membrane+trigger+the+release+of+endocytosed+granzyme+B+into+the+cytosol+of+target+cells.">Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.</a> Nat Immunol. 12, 770-777 (2011).</li><li>Thomas, M. 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=Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins.">Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.</a> J Immunol. 192, 5390-5397 (2014).</li><li>Jacquemin, 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=Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis.">Granzyme B-induced mitochondrial ROS are required for apoptosis.</a> Cell death and differentiation. , (2014).</li><li>Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+recombinant+granzyme+A+zymogen+and+its+activation+by+the+cysteine+proteinase+cathepsin+C.">Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.</a> The Journal of biological chemistry. 271, 9281-9286 (1996).</li><li>Hagn, M., Sutton, V. R., Trapani, 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=A+colorimetric+assay+that+specifically+measures+Granzyme+B+proteolytic+activity:+hydrolysis+of+Boc-Ala-Ala-Asp-S-Bzl.">A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.</a> Journal of visualized experiments : JoVE. , e52419 (2014).</li><li>Huang, M. T., Gorman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+increase+efficiency+of+RNA+3'+processing+and+accumulation+of+cytoplasmic+RNA.">Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.</a> Nucleic Acids Res. 18, 937-947 (1990).</li><li>Luthman, H., Magnusson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+polyoma+DNA+transfection+of+chloroquine+treated+cells.">High efficiency polyoma DNA transfection of chloroquine treated cells.</a> Nucleic Acids Res. 11, 1295-1308 (1983).</li><li>Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+one-stage+method+for+assay+of+enterokinase+activity+by+release+of+radiolabelled+activation+peptides+from+alpha-N-[3H]acetyl-trypsinogen+and+the+effect+of+calcium+ions+on+the+enzyme+activity.">Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.</a> Biochem J. 197, 239-244 (1981).</li></ol>

The authors have nothing to disclose.

GZMAとGZMBの古典と広範囲に研究の役割は、孔形成タンパク質PFN 1によるそれらのサイトゾル送達後、哺乳動物細胞におけるアポトーシスの誘導です。最近、Gzmsの細胞傷害性スペクトルは、細菌9,10、ならびに特定の寄生虫11に、哺乳動物細胞から大幅に広がりました。また、GZMAとGZMBの非古典的、細胞外の機能だけでなく、様々な孤児Gzmsの生物学的意義は依然として不?...

Gzmsは、CTLおよびNK細胞1の専門リソソームに局在高度に相同性セリンプロテアーゼのファミリーです。これらのキラー細胞の細胞傷害性顆粒はまた、排除2,3宛の標的細胞の認識にGzmsと同時にリリースされる膜破壊するタンパク質PFNとGNLYが含まれています。 （ - G、K、MおよびN GZMA）ヒトにおける5 Gzms（GZMA、B、H、KおよびM）、およびマウスでの10 Gzmsがあります。 GZMAとGZMBは最も豊富で広くヒトおよびマウス1で研究されています。しかし、最近の研究は、細胞死経路、ならびに健康と病気4の他、いわゆるオーファンGzmsによって媒介される追加の生物学的効果を検討し始めています。 PFN 5,6によって標的細胞に送達されたときGzmsの最もよく知られている機能は、GZMAおよびGZMBの、特に哺乳動物細胞におけるプログラム細胞死の誘導です。しかしながら、より最近の研究はまた、PFN 7,8によって独立細胞質ゾルデリバリーの、免疫調節および炎症に計り知れない影響を与えGzmsの細胞外効果を実証しました。 Gzmsの細胞質ゾルのエントリの後に効率的に殺された細胞のスペクトルはまた、最近、細菌9,10、さらには特定の寄生虫11に、哺乳動物細胞から広がりました。これらの最近の発見は、Gzmの研究者のための全く新しい分野を開きました。そのため、コスト効率、高収量哺乳動物発現系は、かなりのもの、将来の研究のための方法を容易にします。 天然ヒト、マウス、ラットGzmsが正常にCTLおよびNK細胞株12-14の顆粒画分から精製されています。しかし、我々の手で、このような精製技術の収量は0.1mg / L未満の細胞培養物（未発表の観察及び12）の範囲内です。また、パーソナルプラグインによる汚染のない単一Gzmのクロマトグラフィー分割R Gzmsおよび/ ​​または顆粒中にも存在するタンパク質は、（未発表データと12,14）に挑戦されています。組換えGzms 16は、酵母、昆虫細胞は17とでさえ、このようなHEK 293 18,19などの哺乳動物細胞において、細菌15で製造しました。のみが哺乳動物発現系は、天然の細胞毒性タンパク質と同じ翻訳後修飾を有する組換え酵素を産生する可能性を負います。翻訳後修飾は、エンドサイトーシスによる特異的な取り込みおよび標的細胞20-22内のプロテアーゼの細胞内局在に関与しています。したがって、Gzm発現のためのプラスミド骨格としてpHLsec 23（ラドゥAricescuとイヴォンヌ·ジョーンズの寄贈、オックスフォード、英国の大学）を使用することにより、我々は、高収量のタンパク質産生のための簡単な、時間とコスト効率の高いシステムを構築しましたHEK293T細胞。 pHLsecは、ニワトリΒアクチンプロモーターとCMVエンハンサーを組み合わせました。一緒に、これらの要素はdemonstraテッド種々の細胞株24最強のプロモーター活性。また、プラスミドは、ウサギΒグロビンイントロン、最適化されたKozakおよび分泌シグナル、Lysの-の6xHisタグおよびポリAシグナルが含まれています。インサートは分泌シグナル、適切なN末端 ​​ドメインを欠くタンパク質の最適な発現および分泌の効率を確保するのLys-の6xHisタグ（ 図1）との間で簡便にクローニングすることができます。 EK処理が分泌Gzmsを活性化（アクティブGzmsは、N末端で始まるようにGzmsの発現のために、我々は、エンテロキナーゼ（EK）のサイト（DDDDK）、続いて、ベクターが提供する分泌シグナルと内因性の分泌シグナル配列を置換しアミノ酸配列IIGG 25）。さらに、この方法のために有利なように、HEK293T細胞は、ダルベッコ改変イーグル培地（DMEM）で、低価格媒体中で急速に成長し、コスト効率の高いカルシウムリン酸トランスフェクション法に適しています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

次のセクションでは、この方法を照明するGZMA準備の完全なドキュメントを紹介します。また、正常に精製効率および活性に関して同様の結果を生成する、GZMBとGzmMを精製しました。しかし、それらの後に調製物から、我々は、データの選択したいくつかの作品が表示されます。現在のプロトコルに従って293T細胞からGZMB調製物は、様々な生物学的ア ​​ッセイ系9,29,31-34でそれらの活?...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

哺乳動物細胞におけるヒトグランザイムの高収量およびコスト効率の高い発現システム

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

a-high-yield-cost-efficient-expression-system-human-granzymes

Research

JoVE Journal

Biology

9.7K ビュー.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 

ビデオ: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

生物学

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

ヒト細胞において活性化遺伝子発現のアッセイのダイナミックヒストン修飾のクロマチン免疫沈降（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末試料の絶対量子収率測定

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

哺乳動物細胞における細胞周期の位置の解析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

哺乳類のリンカーヒストンサブタイプの発現解析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...