The authors thank Dara Frank and Carrie Harwood for their generous contribution of strains. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. This research was supported by the National Institutes of Health (R00-GM083147). The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

培養培地、抗生物質の調製、及び2- [2-ニトロ-4-（トリフルオロメチル）ベンゾイル] -1,3-シクロヘキサンジオン（NTBC） <ol><li>適切なボリュームにLBブロス（1％トリプトン、0.5％酵母エキス、H 2 O中 0.25％のNaCl）、アリコートを行います。オートクレーブにより滅菌します。室温で保管してください。 </li><li> 250 mlのフラスコに100 mlのLB寒天培地（1％トリプトン、0.5％酵母エキス、0.25％のNaCl、H 2 O中の1.5％寒天）を作ります。室温でオートクレーブとストアによって滅菌します。寒天プレートに注ぐ前に溶けていることを確認します。 注：LB寒天100mlを含有するフラスコを4板が得られます。 LB寒天培地の量は、アッセイに必要なプレートの数に対応するように変更することができます。 </li><li> PBS（137mMの塩化ナトリウム、2.7のKCl、10mMのの Na 2 HPO 4、2mMのKH 2 PO 4）16ください。室温でオートクレーブまたは濾過し、ストアによって滅菌します。 </li><li>ゲンタマイシンのための抗生物質のストック溶液を準備し、カナマイシン、Dトブラマイシン。 <ol><li> Pの適切な抗生物質ストック濃度を準備100 mg / mlのゲンタマイシン、30 mg / mlのカナマイシン、および10mg / mlのトブラマイシンを含む緑膿菌菌株 。 4℃の水、フィルター滅菌し（0.2ミクロン）、および店舗での抗生物質を溶解します。研究細菌に応じて抗生物質および濃度を変更します。 </li></ol></li><li> NTBCストック溶液を準備します。 DMSOの400μlのNTBC 10mgを溶解します。これは、75.9 mMのNTBCの濃度が得られます。 -20℃で保存NTBC原液。必要に応じて室温でソリューションを解凍します。 注：NTBCの異なるソースは、溶解度の差があります。メーカーの推奨に基づいて、NTBCを溶解し、それに応じてステップ1.5を調整するためにする適切な車両を決定します。 </li></ol>細菌株の2 NTBC滴定<ol><li>テストされる株の一晩培養を設定します。 16×150 mmの試験管に2 mlのLBブロスを加えますS（1株あたり1）と、各株から1に単離したコロニーを接種します。空気インキュベーター中で組織培養回転子に通気しながら37℃で一晩インキュベートします。 </li><li>次の日は、LBブロス中でNTBCの滴定を準備します。異なる株がNTBCに対する感受性の違いを持っ​​ているので、0から900μMNTBCの初期範囲を使用してください。 <ol><li> 1株あたり4〜5本の試験管（16×150ミリメートル）に1ミリリットルのLBブロスを追加します。 </li><li>濃度の範囲内で試験管（16×150ミリメートル）にNTBC原液（75.9 mm）を追加します。 LBブロスの1mlに追加するには、NTBCの濃度とそれに対応する株式ボリュームについては、表1を参照してください。 </li></ol></li><li>一晩培養物のOD 600を測定します。培地中に存在pyomelanin排除するためにOD 600読み取りを行う前に、文化を洗ってください。 <ol><li> 2分間16,000×gで微量に1mlの培養物を遠心分離することによって培養液を洗います。上清を除去し、持つ任意の緩くペレット化した細胞ピペットとは、1ミリリットルのLB固体細胞ペレットを再懸濁</li></ol></li><li> OD 600 0.05で滴定チューブを接種します。チューブに接種するために必要な洗浄文化の量を計算します。 注：pyomelaninが存在してはならないので、予防接種のための洗浄の文化を使用してください。 </li><li>空気インキュベーター中で組織培養回転子を用いて通気しながら37℃で約24時間滴定チューブをインキュベートします。 </li><li>滴定チューブを撮影し、MICおよび酸化ストレスアッセイのために使用するNTBCの量を決定するために、内および系統間で色素産生を比較します。無細胞培養上清中pyomelaninの量を決定し、細胞密度を決定するために、OD 600測定値を使用します。 注：細胞の培養上清中のpyomelaninのOD 600比は、NTBCで処理した後pyomelanin生産の違いを定量化するために計算することができます。 </li></ol> 3.抗生物質の最小Inhibi96ウェルプレートでトリー濃度（MIC）アッセイ<ol><li>し、NTBCないLBにテストされる株の一晩培養を設定します。 注：このプロトコルは、300μMのNTBCの代表レベルを用いて説明します。使用されるNTBCの適切なレベルは、ステップ2.6で決定されます。 <ol><li> 2ミリリットルのLBに300μMのNTBCを追加無NTBC条件の2ミリリットルのLBにビヒクル（DMSO）の同等のボリュームを追加します。 </li><li>滅菌爪楊枝を使用して、細菌のつの単離されたコロニーをチューブに接種。 NTBCと1つの培養し、各株についてNTBCせずに1つの培養があります。空気インキュベーター中で組織培養回転子を用いて通気しながら37℃で一晩インキュベートします。 </li></ol></li><li>次の日、MICアッセイを設定するためのLB + NTBCし、LB + DMSOマスターソリューションを作ります。接種物は、300μMの最終濃度を得、追加されたときに、これは2倍に希釈されるように、600μMの濃度でNTBCを追加します。 <ol><li> 1 antibiをテストするには1株について、耳、2ミリリットルのLBに600μMのNTBCを追加し、NTBCマスター溶液を作製するために混ぜます。 2ミリリットルのLBにビヒクル（DMSO）の等量を追加し、何のNTBCマスター溶液を作成しないように混ぜます。抗生物質の原液を作成するだけでなく、96ウェルプレート中の希釈系列を設定するため、これらのソリューションを使用してください。 注：マスター溶液製剤は、ピペッティングの誤差を考慮して余分な溶液が得られます。試験した抗生物質および株の数に応じて必要に応じてマスターソリューションは拡大または縮小することができます。 </li></ol></li><li> LB + NTBCまたはLB + DMSOマスターソリューションの抗生物質溶液を調製します。 注：これらの溶液中の抗生物質濃度は、二重の最終的な所望の濃度にする必要があります。十分な溶液を、96ウェルプレートの4ウェルに100μlのを転送するようになされるべきです。 <ol><li> 64μgの/ mlのゲンタマイシン+/- NTBC原液を準備します。この溶液を作製するために、450にゲンタマイシン株式（100 mg / mlの）の0.288μlを添加しますμlのLB + NTBCまたはLB + DMSOマスターソリューション。 注：Pのゲンタマイシンの最大濃度緑膿菌 PAO1は、32μgの/ mlです。 </li><li>カナマイシン+/- 256 / mlのでNTBC原液を作ります。この溶液を作製するために、450μlのLB + NTBCまたはLB + DMSOマスターソリューションにカナマイシンストック（30 mg / mlの）の3.84μlを添加します。 注：Pのカナマイシンの最大濃度緑膿菌 PAO1は、128μgの/ mlです。 </li><li>トブラマイシン+/- 8 / mlのでNTBC原液を準備します。この溶液を作製するために、450μlのLB + NTBCまたはLB + DMSOマスターソリューションにトブラマイシンストック（10mg / ml）の0.36μlを添加します。 注：Pの緑膿菌 PAO1、トブラマイシンの最大濃度は、4 / mlのです。 注：細菌を試験するために、抗生物質との濃度を調整することができます。 </li></ol></li><li> 96ウェルプレートの4つのウェルにそれぞれ2倍の抗生物質溶液100μlを追加します。 examplの行Aにこれらのソリューションを配置しますeは、ゲンタマイシンがA4を通じてA1に配置する必要があり、カナマイシンはA8を通じてA5に配置する必要があり、およびトブラマイシンはA12を通じてA9に配置する必要があります。セットアップ、96ウェルプレートの図については、図1Aを参照してください。 注：複数の抗生物質は、一枚のプレートで試験することができるが、唯一の1株は他の株からの交差汚染の可能性を排除するために、プレートごとにテストする必要があります。 </li><li> 96ウェルプレートのHを通じて行BにLB + NTBCまたはLB + DMSOマスター溶液50μlを追加します。一方のプレートは、LB + NTBCであることを確認し、一方のプレートは、LB + DMSOです。 図1Aを参照してください。 <ol><li> LB + NTBCにおける抗生物質のためのLB + NTBCを使用してください。 LB + DMSO中の抗生物質のためのLB + DMSOを使用してください。 </li></ol></li><li>マイクロピペットを使用すると、ピペットチップを変更、Bのミックスにソリューションを行ごとにAから溶液50μlを転送することにより、抗生物質の2倍連続希釈を行い、のためのC.繰り返しを行ごとにBから溶液50μlを転送remainingの行。行Gを希釈した後、その行から溶液50μlを除去し、廃棄します。細菌の増殖のための抗生物質のないコントロールとしてH列を使用してください。 図1Bを参照してください 。 注：Gを通じて行Aの各ウェルは現在の2倍でLB + NTBCまたはLB + DMSO中の抗生物質、50μlの最終的な所望の濃度が含まれています。 H列は、抗生物質なしでLB + NTBCまたはLB + DMSOが含まれています。 </li><li>一晩培養物のOD 600を測定します。培地中に存在pyomelaninを排除するためにOD 600読み取りを行う前に、すべての文化を洗ってください。 <ol><li> 2分間16,000×gで微量に1mlの培養物を遠心分離することによって培養液を洗います。ピペットで上清を除去し、1mlのLBに細胞ペレットを再懸濁</li></ol></li><li> LBの中で2.75x10 5 CFU / mlに一晩培養を希釈 注：1 OD 600ユニットは、Pの 1×10 9 CFU / mlと同等であると仮定緑膿菌 。 CFU / mlの変換へのODは、Dすることができます他の細菌でifferent。 </li><li>適切なウェルに希釈した細菌培養物の50μlのを追加します。 注：NTBCで増殖させた培養物は、DMSO中で増殖させ、培養物NTBCを含むウェルに添加されるべきではDMSOを含有するウェルに添加されるべきです。 図1Bを参照してください 。 <ol><li>各菌株と抗生物質濃度のための3つのウェルに細菌を追加します。細菌汚染のための対照として機能する第四ウェルにLB50μlのを追加します。 図1Bを参照してください 。 </li><li>井戸を接種するために、マルチチャンネルピペットを使用してください。近隣の井戸の汚染を防止するために、接種材料を追加する際にピペットチップは、ウェルの底近くにあることを確認してください。 注：ウェルに細菌培養を追加すると、抗生物質とNTBC濃度2倍に希釈します。 </li></ol></li><li>パラフィルムを有する96ウェルプレートを覆い、37℃で約24時間インキュベートします。空気インキュベーター中、静的96ウェルプレートをインキュベートします。 </li><li>調べウェル中の細菌増殖のためのプレート。 MICには、細菌の増殖を、各株のすべての3つの複製のために見ていないされている抗生物質の最低濃度です。 <ol><li>視覚的な成長のためにプレートを調べたり、OD 600に設定し、プレートリーダーを用いて読み取ります。 </li></ol></li></ol>酸化ストレス応答4.スポットプレートアッセイ<ol><li>ステップ3.1で説明したようにNTBCとないLB中でテストされる株の一晩培養を設定します。 </li><li>翌日、酸化的ストレッサーとしてH 2 O 2を含有する LB寒天プレートを調製します。 0〜1 mMのからのH 2 O 2濃度の範囲は、良い出発点です。 <ol><li> LB寒天フラスコを溶かします。室温で約50℃に冷却するメディア。 </li><li>所望の濃度の冷却媒体に直接H 2 O 2 を追加します 。スワールフラスコを混合します。 H 2 O 2の濃度は 、表2を参照そして濃縮し、H 2 O 2のボリュームが追加されます。これらの値は、LB寒天100mlのに基づいています。 </li><li>すぐにH 2 O 2を添加した後 、プレートを注ぎ、気泡を除去するために表面を火炎。収率はLB寒天の100ミリリットル当たり4板です。マーク・H 2 O 2濃度でプレート。 </li><li>プレートから余分な水分を除去するために、30分間実行しているファンとの生物学的な流フードで明らかになったプレートを置きます。 注：プレート、それらが用意されている同じ日に使用します。そうしないと、一貫性のないデータになることがあります。 注：例えば、パラコートなどの酸化ストレスは、このアッセイにおけるH 2 O 2の代わりに用いることができます。他の酸化ストレスのために使用される濃度は、H 2 O 2のために使用されるものと異なってもよいです。 </li></ol></li><li>洗浄ステップ3.7で説明したように一晩培養のOD 600を測定します 。 </li><li>すべて一晩のcuのOD 600を正規化テストされている株のセットの最低値にltures。P.緑膿菌は、一般的に 、LB + NTBCまたはLB + DMSO中で一晩増殖させ、約2.5のOD 600を持っています。 <ol><li> 1ミリリットルの全容量で最低のOD 600に文化を希釈するために必要な培養物の容量を決定します。例えば、培養物がOD 3の600を有しており、株のセットのための最小のOD 600が 2.5であり、次の計算実行する場合：（2.5）（1ミリリットル）=（3）（X）。 X = 0.833ミリリットル。文化の0.833ミリリットルをマイクロチューブ内に配置されます。 </li><li> 1mlに培養容量をもたらすために必要なLB + NTBC（300μM）またはLB + DMSOの量を計算します。ステップ4.4.1の例では、LB + NTBCまたはLB + DMSOの量は0.167ミリリットル（ - 0.833ミリリットル文化1ミリリットル総容量）になる培養液に添加。 LB + NTBCし、LB + DMSOのストック溶液は、すべての株を希釈するために必要な量に基づいて、これらの希釈液に使用するようにしてください。 </li><li>文化とLB + Nを混ぜますボルテックスによってTBCまたはLB + DMSO。 </li></ol></li><li> NTBCまたはDMSOの一定濃度で培養を維持するために、PBS + NTBCまたはPBS + DMSO中の正規化された一晩培養の10倍連続希釈を行います。 <ol><li> PBS + NTBCし、PBS + DMSOのストック溶液を作ります。 1株についての希釈液の1セットでは、720μLの総容量を得るために、PBSで300μMのNTBCまたはDMSOの等量を混ぜます。テストされてどのように多くの株に応じて上下にこれらの株式をスケール。 </li><li> 10 -7の連続希釈10〜-1のラベルマイクロチューブ。適切なチューブにPBS + NTBCまたはPBS + DMSO90μlのを追加します。 LB + NTBCで成長した株について、PBS + NTBCを使用し、LB + DMSO中で成長した株をPBS + DMSOを使用しています。 </li><li>適切な10 -1希釈チューブに文化の10μlのを追加します。ボルテックスで混合し、10 -2希釈チューブに10 -1希釈液10μlを移します。すべての希釈はperfoされるまで繰り返しますrmed。希釈液の間にピペットチップを変更します。 </li></ol></li><li>各株の重複でLB + H 2 O 2のプレートに10 -7希釈液を10〜-3のスポット5μlの。スポットがメッキされている場合は、ほとんどが少なくとも希（10 -7〜10 -3）に希釈から1ピペットチップを使用してください。液体がプレートに乾燥するまでプレートを傾けたり、傾けたりしないでください。 </li><li>歪みに応じて、37℃（空気インキュベーター）で24〜48時間、プレートをインキュベートします。 注：P。緑膿菌 PAO1は、インキュベーションの24時間後に、LBの良いサイズのコロニーを持つことになります。それらがコロニーをPAO1とほぼ同じ大きさになるまで株を培養します。 </li><li> transluminator上にCCDカメラを用いてプレートを撮影。必要に応じて、同じサイズとは対照的と作物のために写真を編集します。酸化ストレスに対する感受性の変化を決定するために各スポットにコロニー数をカウントします。 </li></ol>

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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=Genome+mosaicism+is+conserved+but+not+unique+in+Pseudomonas+aeruginosa++from+the+airways+of+young+children+with+cystic+fibrosis.">Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa from the airways of young children with cystic fibrosis.</a> Environ Microbiol. 5, 1341-1349 (2003).</li><li>Sanchez-Amat, A., Ruzafa, C., Solano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+tyrosine+degradation+in+Vibrio+cholerae++The+strain+ATCC+14035+as+a+prokaryotic+melanogenic+model+of+homogentisate-releasing+cell.">Comparative tyrosine degradation in Vibrio cholerae The strain ATCC 14035 as a prokaryotic melanogenic model of homogentisate-releasing cell.</a> Comp Biochem Physiol B Biochem Mol Biol. 119, 557-562 (1998).</li><li>Hunter, R. C., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Putative+ABC+Transporter,+HatABCDE,+Is+among+Molecular+Determinants+of+Pyomelanin+Production+in+Pseudomonas+aeruginosa.">A Putative ABC Transporter, HatABCDE, Is among Molecular Determinants of Pyomelanin Production in Pseudomonas aeruginosa.</a> J Bacteriol. 192, 5962-5971 (2010).</li><li>Ketelboeter, L. M., Potharla, V. Y., Bardy, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NTBC+treatment+of+the+pyomelanogenic+Pseudomonas+aeruginosa++isolate+PA1111+inhibits+pigment+production+and+increases+sensitivity+to+oxidative+stress.">NTBC treatment of the pyomelanogenic Pseudomonas aeruginosa isolate PA1111 inhibits pigment production and increases sensitivity to oxidative stress.</a> Curr Microbiol. 69, 343-348 (2014).</li><li>Kavana, M., Moran, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+(4-Hydroxyphenyl)pyruvate+Dioxygenase+with+the+Specific+Inhibitor+2-[2-Nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.">Interaction of (4-Hydroxyphenyl)pyruvate Dioxygenase with the Specific Inhibitor 2-[2-Nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.</a> Biochemistry. 42, 10238-10245 (2003).</li><li>Andrews, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+minimum+inhibitory+concentrations.">Determination of minimum inhibitory concentrations.</a> J Antimicrob Chemother. 48, 5-16 (2001).</li><li>Nikodinovic-Runic, J., Martin, L. B., Babu, R., Blau, W., O'Connor, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+melanin-overproducing+transposon+mutants+of+Pseudomonas+putida+F6.">Characterization of melanin-overproducing transposon mutants of Pseudomonas putida F6.</a> FEMS Microbiol Lett. 298, 174-183 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Roy-Burman, 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=Type+III+protein+secretion+is+associated+with+death+in+lower+respiratory+and+systemic+Pseudomonas+aeruginosa+infections.">Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections.</a> J Infect Dis. 183, 1767-1774 (2001).</li><li>Jacobs, M. 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=Comprehensive+transposon+mutant+library+of+Pseudomonas+aeruginosa.">Comprehensive transposon mutant library of Pseudomonas aeruginosa.</a> Proc Natl Acad Sci USA. 100, 14339-14344 (2003).</li><li>Youngchim, S., Pornsuwan, S., Nosanchuk, J. D., Dankai, W., Vanittanakom, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanogenesis+in+dermatophyte+species+in+vitro++during+infection.">Melanogenesis in dermatophyte species in vitro during infection.</a> Microbiology. 157, 2348-2356 (2011).</li><li>Khajo, 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=Protection+of+Melanized+Cryptococcus+neoformans+Lethal+Dose+Gamma+Irradiation+Involves+Changes+in+Melanin's+Chemical+Structure+and+Paramagnetism.">Protection of Melanized Cryptococcus neoformans Lethal Dose Gamma Irradiation Involves Changes in Melanin's Chemical Structure and Paramagnetism.</a> PLoS ONE. 6, e25092 (2011).</li><li>Hancock, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hancock Laboratory Methods. , (2015).</li></ol>

The authors declare that they have no competing financial interests.

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates12 (Figure 2). The...

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress1-4 and binds a variety of compounds, including aminoglycoside antibiotics5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)1,9 or through imbalances in the various enzymes in the pathway10. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin11. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)12, which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway13. Hpd is required for the formation of homogentisate, and therefore pyomelanin11. 
We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.
The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa12. Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction. 
The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent12. Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.
Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions14. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.
Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time1, or oxidative stress disc diffusion assays15. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H2O2 and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td>
			<td>Sigma-Aldrich</td>
			<td>SML0269-50mg</td>
			<td>Also called nitisinone.&#160; Soluble in DMSO.</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td>30 wt. % in H2O.&#160; Stabilized.</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gold Bio</td>
			<td>G-400-100</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014-100MG</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
	</tbody>
</table>

methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

NTBC滴定 NTBCはP.でpyomelanin産生を減少させることができた場合にNTBC滴定を決定するために使用されました緑膿菌 、また、追加のアッセイにおける使用のためのpyomelanin生産性を排除または低減されるNTBCの濃度を特定します。そこ異なる複製で生産pyo​​melaninのレベルの変化であるが、一般的な傾向は一定のままであってもよいです。 NTBC滴定アッセイはまた?...

Watch this Scientific Journal Video about Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress at JoVE.com

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

細菌Pyomelanin産生を阻害し、酸化ストレスに対する感受性の増加に対応を決定するための方法

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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Research

JoVE Journal

Immunology and Infection

8.1K ビュー.  University of Wisconsin Milwaukee. Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

ビデオ: Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

免疫・感染学

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

定性的にオーバーレイアッセイを使用すると、肺炎球菌バクテリオシンに対する細菌の産生と感度を測定します

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

マラリアに一塩基多型のジェノタイピングを溶融高解像度の感度を上げる方法

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

in vitroおよびin vivoモデルでは血管壁の下でフローの状況に細菌付着を研究するために、

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

血液処理のための迅速な方法は、ヒト血液中の血漿ペプチドレベルの収量を増やすには

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

ヒトの過剰発現と精製<em&gt;シス</em&gt; - プレニルトランスフェラーゼ<em&gt;大腸菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

代謝のマッピング: 定量的酵素組織化学および組織化学細胞や組織における脱水素酵素の活性を決定するには

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...