Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

この作品は、韓国研究所の計画と評価における食品・農業・林業 (IPET) 農水省・食品・農村情勢 (マフラによって資金を供給、農業バイオ技術開発プログラムを通じて技術によって支えられました。) (116159-02-2-WT011) 生命科学部と bk 21 プラス、韓国大学バイオ テクノロジー。

1. 媒体、化合物、および試薬の調製
<ol><li>B16F10 成長完全培地を準備します。サプリメント ダルベッコ イーグル培地 (DMEM) 10% 牛胎児血清 (FBS) と 1% ペニシリン/ストレプトマイシン (害虫) に変更。<ol>
<li>500 mL の完全培地を作るため、FBS と 1 L 滅菌ガラス瓶の中の害虫 5 mL 50 ml ダルベッコ変法イーグル培地 (DMEM) 445 mL を混ぜます。軽く混合後、新しい滅菌ガラス瓶と 4 ° C で、ストアに 0.2 μ m ...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protective+role+of+melanin+against+UV+damage+in+human+skin.">The protective role of melanin against UV damage in human skin.</a> Photochemistry and Photobiology. 84 (3), 539-549 (2008).</li><li>Galvan, I., 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=Melanin+chemistry+and+the+ecology+of+stress.">Melanin chemistry and the ecology of stress.</a> Physiological and Biochemical Zoology. 88 (3), 352-355 (2015).</li><li>Burger, P., Landreau, A., Azoulay, S., Michel, T., Fernandez, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+whitening+cosmetics:+Feedback+and+challenges+in+the+development+of+natural+skin+lighteners.">Skin whitening cosmetics: Feedback and challenges in the development of natural skin lighteners.</a> Cosmetics. 3 (4), 36 (2016).</li><li>Cooksey, C. 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=Evidence+of+the+indirect+formation+of+the+catecholic+intermediate+substrate+responsible+for+the+autoactivation+kinetics+of+tyrosinase.">Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase.</a> Journal of Biological Chemistry. 272 (42), 26226-26235 (1997).</li><li>Ando, H., Kondoh, H., Ichihashi, M., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+identify+inhibitors+of+melanin+biosynthesis+via+the+quality+control+of+tyrosinase.">Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase.</a> Journal of Investigative Dermatology. 127 (4), 751-761 (2007).</li><li>Gillbro, J. M., Olsson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+melanogenesis+and+mechanisms+of+skin-lightening+agents+-+existing+and+new+approaches.">The melanogenesis and mechanisms of skin-lightening agents - existing and new approaches.</a> International Journal of Cosmetic Science. 33 (3), 210-221 (2011).</li><li>Passeron, T., Coelho, S. G., Miyamura, Y., Takahashi, K., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+and+in+situ+hybridization+in+the+study+of+human+skin+melanocytes.">Immunohistochemistry and in situ hybridization in the study of human skin melanocytes.</a> Experimental Dermatology. 16 (3), 162-170 (2007).</li><li>Lee, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+inhibitions+of+lemon+balm+(Melissa+officinalis)+ethanolic+extract+on+melanogenesis+in+B16-F1+murine+melanocytes:+inhibition+of+tyrosinase+activity+and+its+gene+expression.">Dual inhibitions of lemon balm (Melissa officinalis) ethanolic extract on melanogenesis in B16-F1 murine melanocytes: inhibition of tyrosinase activity and its gene expression.</a> Food Science and Biotechnology. 20 (4), 1051-1059 (2011).</li><li>Jun, H. 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=p-Coumaric+acid+inhibition+of+CREB+phosphorylation+reduces+cellular+melanogenesis.">p-Coumaric acid inhibition of CREB phosphorylation reduces cellular melanogenesis.</a> European Food Research and Technology. 235 (6), 1207-1211 (2012).</li><li>Jun, H. 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=Dual+inhibition+of+&#947;-oryzanol+on+cellular+melanogenesis:+inhibition+of+tyrosinase+activity+and+reduction+of+melanogenic+gene+expression+by+a+protein+kinase+a-dependent+mechanism.">Dual inhibition of &#947;-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase a-dependent mechanism.</a> Journal of Natural Products. 75 (10), 1706-1711 (2012).</li><li>Choi, Y. 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=Effects+of+the+isoflavone+puerarin+and+its+glycosides+on+melanogenesis+in+B16+melanocytes.">Effects of the isoflavone puerarin and its glycosides on melanogenesis in B16 melanocytes.</a> European Food Research and Technology. 231 (1), 75-83 (2010).</li><li>Cho, B. R., Jun, H. J., Thach, T. T., Wu, C., Lee, 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=Betaine+reduces+cellular+melanin+content+via+suppression+of+microphthalmia-associated+transcription+factor+in+B16-F1+murine+melanocytes.">Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes.</a> Food Science and Biotechnology. 26 (5), 1391-1397 (2017).</li><li>Overwijk, W. W., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B16+as+a+mouse+model+for+human+melanoma.">B16 as a mouse model for human melanoma.</a> Current Protocols in Immunology. , (2001).</li><li>Virador, V. M., Kobayashi, N., Matsunaga, J., Hearing, V. 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+standardized+protocol+for+assessing+regulators+of+pigmentation.">A standardized protocol for assessing regulators of pigmentation.</a> Analytical Biochemistry. 270 (2), 207-219 (1999).</li><li>D'Mello, S. A. N., Finlay, G. J., Baguley, B. C., Askarian-Amiri, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+melanogenesis.">Signaling pathways in melanogenesis.</a> International Journal of Molecular Sciences. 17 (7), 1144 (2016).</li><li>Hou, L., Panthier, J. J., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+the+neural+crest-derived+melanocyte+lineage:+interactions+between+KIT+and+MITF.">Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF.</a> Development. 127 (24), 5379-5389 (2000).</li><li>Hedley, S. J., Gawkrodger, D. J., Weetman, A. P., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-MSH+and+melanogenesis+in+normal+human+adult+melanocytes.">&#945;-MSH and melanogenesis in normal human adult melanocytes.</a> Pigment Cell Research. 11 (1), 45-56 (1998).</li><li>Hu, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+evaluation+of+melanin+content+and+production+of+pigment+cells+in+vitro.">Methodology for evaluation of melanin content and production of pigment cells in vitro.</a> Photochemistry and Photobiology. 84 (3), 645-649 (2008).</li><li>Carriel, V. 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=A+novel+histochemical+method+for+a+simultaneous+staining+of+melanin+and+collagen+fibers.">A novel histochemical method for a simultaneous staining of melanin and collagen fibers.</a> Journal of Histochemistry &amp; Cytochemistry. 59 (3), 270-277 (2011).</li></ol>

培養メラノサイトを用いた試験化合物の色素脱失活性を評価するためのプロトコルを提案します。代表的な結果は、アルブチン、チロシナーゼの活性と細胞のメラニン コンテンツを抑制するチロシナーゼ阻害剤の色素脱失の効果を示した。これらのメソッドは抗メラニン活性研究で広く使用されます。これらの試金を使用して、我々 はまた、正常に B16F10 細胞のメラニン合成阻害効果が過去...

メラニンは、生理学、病理学、皮膚、目、脳1などいくつかの臓器の毒物学で重要な役割を果たしています。メラニンの主要な機能は、写真審査と生化学的な効果です。メラニンは uv (紫外線) 放射、光の短波長の光で増加した吸収率と同様、近赤外、可視光を吸収します。したがって、メラニンは、可視光や紫外線放射2によるダメージから組織を保護します。メラニンの酸化防止剤、金属および他の有毒化学物質に親和性を持ってしたがって、それは、化学的酸化ストレス3から組織を保護できます。ただし、メラニンの過剰生産は、皮膚の問題を引き起こします。
肌や虹彩のメラニンの質と量は、アイリスと肌の色の最も重要な要因です。個人があります異なる肌の色の設定;明るい皮膚色を好む他、日焼けした肌、賛成します。これらの消費者プロファイルに応じて化粧品色素脱失を支持された肌の色4の個々 の市場を満たすために開発されています。したがって、科学的に、実質的に、色素脱失、抗メラニン活性研究が重要です。
メラニンは、メラノサイトでの酵素および自発の化学反応の一連のメラニン生合成の複雑なプロセスです。1 つの色素は約 36 ケラチノ サイトに囲まれ、メラノサイトは、メラニン合成工場隣接のケラチノ サイトへの彼らの製品を配布します。皮膚、近隣の樹状突起を介して表皮ケラチノ サイトにメラニン色素の生産し、メラニン細胞の melanosomal のコンパートメントに格納されているで運ばれています。
L-チロシンをメラニン形成と酵素チロシナーゼの初期基板は 3, 4-ジヒドロキシフェニルアラニン (DOPA) にし、dopaquinone に L-チロシンに変換 2 つの連続した反応を触媒する提供しています。これらの反応メラニン形成5,6の率制限ステップであります。したがって、色素脱失活動は、まず直接細胞のチロシナーゼ活性を評価することによって測定できます。ドーパと孵化させるメラニン細胞エキスのチロシナーゼが含まれているようにとサンプルで生産 dopaquinone 475 に吸光光度法によって測定できる nm。値は、サンプル、およびコントロールと比較して少ない dopaquinone 形成における色素脱失活動結果と物質のタンパク質の濃度によって正規化されます。
第二に、培養メラノサイトでメラニンを直接測定することにより色素脱失活動を定量化することができます。試験材料とセルを扱う後、メラニンがアルカリ条件下で抽出され、メラニン コンテンツを 400 で吸光光度法で定量 nm。色素脱失エージェントはコントロール7より低いメラニン コンテンツになります。
最後に、フォンタナ ・ マッソン メラニン染色とそれに続く画像解析による色素脱失活動を定量化することができます。フォンタナ ・ マッソン染色にメラニン顆粒が見える黒い金属状態にアンモニア硝酸銀を減らすし、細胞顕微鏡画像の黒の領域は、メラニンの量を表します。
色素脱失のエージェントは、通常物質の活動が有効であることを確認これらの 3 つの方法の一貫性と同等の結果を与えます。また、メラニン色素脱失活性を調べるテスト物質に応答のキー遺伝子そして蛋白質の発現を測定する有用な場合があります。チロシナーゼ、に加えてチロシナーゼ関連タンパク質 (TRP-1) と dopachrome tautomerase (TRP 2) がメラニン形成8の重要な酵素です。メラニン形成とその遺伝子・ タンパク質発現レベルの定量化のマスター調節因子である転写因子小眼球関連転写因子 (MITF) またはプロモーターの活性測定法は色素脱失活性を評価するためにも使用できます。

<table>
	<tbody>
		<tr>
			<td>3,4-Dihydroxy-l-phenylalanine</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma</td>
			<td>#320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Arbutin</td>
			<td>Fluka</td>
			<td>#10960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Yakuri Pure Chemicals</td>
			<td>#16223</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>American MasterTech</td>
			<td>AHG0226</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Samchun chemical</td>
			<td>H0255</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bio basic Canada Inc.</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T.Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duksan pure chemicals</td>
			<td>#81</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear fast red</td>
			<td>Merck</td>
			<td>100121</td>
			<td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>J.T. Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate pentahydrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#2163</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Bio Basic Canada</td>
			<td>TB0196</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Union Carbide</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosinase from mushroom</td>
			<td>Sigma</td>
			<td>T3824</td>
			<td>25 KU</td>
		</tr>
	</tbody>
</table>

quantification of hypopigmentation activity in vitro

本研究では、色素脱失活性の in vitro の定量化のための実験方法を示します。メラニンは、メラノサイトの主要な色素は、複数の細胞および環境要因への応答で合成されます。メラニンは紫外線のダメージから皮膚細胞を保護、生物物理学的および生化学的な機能を持っています。過剰生産やメラノサイトでメラニンの蓄積は、そばかすやシミ、肝斑、ほくろなどの皮膚の問題を引き起こす可能性が。したがって、メラニン色素脱失エージェントのコントロールは重要な臨床的または化粧品ニーズを持つ個人です。メラニンは、メラノサイト メラニンに強く影響を外因性と呼ばれる複雑な生化学的過程とホルモン、炎症、年齢、紫外線暴露などの内的因子のメラノソームで主に合成されています。我々 は化学物質やメラノサイトの天然物質の色素脱失アクティビティを決定する 3 つの方法を記述する: 1) 細胞のチロシナーゼ活性・ 2) メラニン コンテンツ イメージ 3) 染色および定量化の細胞メラニンの測定解析。
メラニン形成、チロシナーゼは 3, 4-ジヒドロキシフェニルアラニン (L-DOPA) にし、dopaquinone に L-チロシンに変換する律速段階を触媒します。したがって、チロシナーゼの阻害、主色素脱失メカニズムです。培養メラノサイトでチロシナーゼ活性は基板として L-DOPA を追加し、dopaquinone の生産を吸光光度法による測定で定量化できます。メラニンは、メラニン コンテンツを定量化することで測定できます。NaOH でメラニン含有細胞分画を抽出し、メラニンが光光度法による定量化されます。最後に、次のメラニンのフォンタナ ・ マッソン染色画像解析によるメラニン コンテンツを定量化することができます。これらの試験管内アッセイの結果が常に人間の皮膚で再現することができません、これらのメソッドは、潜在的な色素脱失の活動を識別する最初のステップとして特に広くメラニン形成研究で使用します。これらのメソッドは、メラニン細胞の活性・成長・分化を評価するためにも使用できます。3 つの異なる方法で一貫した結果では、効果の有効性を確認します。

アルブチン、抗メラニンの色素脱失活動の代表の結果は、B16F10 メラノサイトは以下の化合物します。図 1 aは、そのアルブチンが車両処理制御に比べ細胞のチロシナーゼ活性を有意に抑制を示しています。同様に、刺激アルブチンのメラニン コンテンツ コントロール (図 1 b) と比較して著しく減った。フォンタナ ・ マッ?...

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Quantification of Hypopigmentation Activity In Vitro

化学物質の in vitro の色素脱失活性を評価するための 3 つの実験的方法を述べる: 1) 細胞のチロシナーゼ活性および 2) メラニン コンテンツ、および 3) メラニン細胞のメラニン色素染色と画像解析による測定の定量化。

培養色素脱失活動の定量化

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

この方法は、抗メラノジェニック活性を有する機能性化粧品および皮膚剤の開発に有用であり得る。それは実行する必要があり、結果はすぐに得ることができます。さらに、この方法は、研究者のために有用であり得る, 効果や化合物を同定または調査したり、前起性または原発性の活性を調査したい人.チロシナーゼ活性を測定するには、細胞培養インキュベーターで24時間完全培地中のウェル当たり4番目の細胞にB16-F10メラニシドを5回10回から4番目の細胞に播種することから始める。翌日、各ウェルの上澄み物を、作りたての試験化合物と阻害剤コントロールまたは陰性対照で補ったDMEMに置き換え、プレートをさらに72時間インキュベーターに戻します。治療の終わりには、井戸あたり300マイクロリットルの冷たいPBSで井戸を2回すすいで、プレートを氷の上に置きます。次に、セルスクレーパーを使用して各ウェルに300マイクロリットルのライシスバッファーを加え、5分後に細胞ライセートを収集します。得られた細胞粒子懸濁液を個々の1.5ミリリットルのマイクロ遠心分離管に移す。そして、ライセートを14、500rpmで2秒間3回均質化し、細胞間チロシナーゼを放出する。遠心分離によって細胞の破片をペレットし、氷上の個々の1.5ミリリットル遠心管に上清を移す。クリア96ウェルポリスチレンマイクロプレートの個々のウェルに各上清の70マイクロリットルを割り当て、上清の各ウェルにチロシナーゼ基質溶液の140マイクロリットルを追加します。●プレートを優しく振って、ウェル内容物を混ぜます。その後、2時間摂氏37度でプレートをインキュベートします。そしてマイクロプレートリーダー上の475ナノメートルでチロシナーゼ活性を測定する。処置した細胞のライセートを収集した後の細胞のメラニン含有量を測定する。遠心分離によってライセートを収集し、新しいチューブに上清を転送します。摂氏60度で1時間のインキュベーションのために各ペレットに1つの通常の水酸化ナトリウムの300マイクロリットルを追加します。その後、溶解した細胞懸濁液の遠心分離が続いた。その後、上清の200マイクロリットルを96ウェルプレートの各ウェルに移します。そして、400ナノメートルの波長でマイクロプレートリーダー上のメラニン内容物を測定する。フォンタナ・マッソン染色の場合、処理した細胞を300マイクロリットルの冷たいPBSで2回洗浄し、摂氏4度で1時間10%ホルマリンの200マイクロリットルで細胞を固定します。濃厚な細胞を蒸留水ですすい、58~68°Cの200マイクロリットルを各井戸に加えます。摂氏37度で1時間のインキュベーション、または細胞が黄色褐色になるまで。染色した細胞を蒸留水でリンスし、その後室温で0.1%の塩化金で2分間インキュベートします。蒸留水で処理した金の塩化物を、200マイクロリットルのチオ硫酸ナトリウム溶液を細胞に加えます。2分後に再び細胞をすすきます。そして、5分間、ウェルあたり200マイクロリットルの核高速赤で細胞にラベルを付けます。次に、染色した細胞を水道水で洗浄してから、光学顕微鏡でイメージングします。しきい値解析の場合は、画像 J で画像を開き、画像、調整、およびカラーしきい値を選択します。Fontana Masson で染色された領域を測定するには、明るさパラメータバーをゼロに設定し、黒または茶色の染色セルがすべて含まれる位置に明るさを調整します。[パーティクルの解析と解析] を選択し、[パーティクルの解析] ウィンドウの [集計] ボックスをオンにし、[問題] をクリックして結果の概要を取得し、データをスプレッドシートにコピーして貼り付けます。アルブチン処理は、制御車両処理細胞と比較して細胞性チロシナーゼ活性を有意に抑制する。同様に、アルブチンで刺激された細胞のメラニン含量は、コントロールに比べて有意に減少する。細胞は治療群間で形態学的に類似しているが、アルブチンは、コントロール処理された細胞と比較して黒色色素の面積を減少させる。この方法は、複合ライブラリーを使用した活動スクリーニングに役立ちます。迅速にテストするサンプルの数百以上があります。また、この方法は、メラニン生成を伴うメカニズムを調べることもできます。生物活性候補化合物が同定された後、生物学的メカニズムまたは商用アプリケーションの研究に対する彼らの証言のために。

quantification-of-hypopigmentation-activity-in-vitro

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

のpH依存性活性の検出<em&gt;エシェリヒア・コリ</em&gt;シャペロンHdeB<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

生化学

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

豊かさと充電転送エシェリヒア属大腸菌の Rna のレベルの定量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

マッピング代謝: 監視組織で直接乳酸脱水素酵素活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

TRNA イソペンテニル トランスフェラーゼ活性を検出する体外の試金

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

多量ホモ測定器と無料、フリー、オープン ソースの明るさ解析ソフトウェアを使用しての体外校正無料定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

ショウジョウバエメラノガスターにおけるクエン酸シンターゼ活性の比色アッセイ

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

カエノルハブ炎エレガンスにおける2-アラチドノイルグリセロールの定量のための過熱基準の合成

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

多重共免疫沈殿を用いたタンパク質相互作用ネットワークダイナミクスの定量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

細胞および組織におけるコエンザイムAの定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

mtDNAの合成と分布のハイスループット定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

著者スポットライト:ミトコンドリアDNA合成と分布のハイスループット画像ベースの定量化  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

ミトコンドリアDNA合成と分布のハイスループット画像ベースの定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...