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

Bu eser Kore Enstitüsü planlama ve değerlendirme için gıda, tarım ve Ormancılık (IPET) Tarım Bakanlığı, gıda ve kırsal işleri (MAFRA tarafından finanse edilen ağrı-Bioindustry teknoloji geliştirme programı aracılığıyla teknolojisi tarafından desteklenen ) (116159-02-2-WT011) ve okul Yaşam Bilimleri ve biyoteknoloji BK21 Plus, Kore Üniversitesi.

1. orta, bileşikler ve Kimyasalları hazırlanması
<ol><li>B16F10 büyüme tam orta hazırlayın. Ek Dulbecco'nın kartal orta (DMEM) ile % 10 fetal sığır serum (FBS) ve % 1 penisilin/streptomisin (zararlı) değiştiren.<ol>
<li>500 mL tam orta yapmak için Dulbecco'nın değiştirilmiş kartal orta (DMEM) 445 mL 50 mL FBS ve haşere 1 L steril cam şişede 5 mL ile karıştırın. Hafifçe karıştırdıktan sonra orta yeni steril cam şişe ve 4 ° C'de sonra mağaza 0.2 µm şişe-üst filtre ile filtre uygula...

<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+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>

Biz kültürlü melanosit kullanarak test bileşiklerin hipopigmentasyon etkinliğini değerlendirmek için protokol sundu. Temsil edici sonuçlar arbutin, Tirozinaz etkinlik ve hücresel melanin içerik inhibe bir Tirozinaz inhibitörü hipopigmentasyon etkisini gösterdi. Bu yöntemler anti-melanogenic aktivite araştırmalarda yaygın olarak kullanılmaktadır. Bu deneyleri kullanarak, biz de başarıyla geçmişte melanogenesis B16F10 hücreleri üzerinde inhibitör etkisi olduğu birçok biyoaktif bileşikler belirl...

Melanin fizyolojisi, patoloji ve toksikoloji cilt, göz ve beyin1de dahil olmak üzere çeşitli organlarının kritik bir rol oynamaktadır. Büyük melanin fotoğraf-tarama ve biyokimyasal etkileri işlevlerdir. Melanin ultraviyole radyasyon, ışığın daha kısa dalga boyları artan emme oranları ile yanı sıra yakın kızılötesi ve görünür ışık emer; Böylece, melanin görünür ışık veya UV radyasyon2neden hasar dokuları korur. Melanin bir antioksidan ve metaller ve diğer toksik kimyasal maddeler için bir ilgi vardır; Bu nedenle, o-ebilmek korumak dokulara kimyasal ve oksidatif stres3. Ancak, aşırı melanin üretimini dermatolojik sorunlarına neden olur.
Miktar ve kalite melanin deride ve iris Iris ve cilt rengini en önemli belirleyicileri vardır. Bireyler farklı cilt renk tercihleri olabilir; diğerleri daha açık cilt renkleri lehine iken bazı tabaklanmış deri, iyilik. Bu tüketici profilleri bağlı olarak hipopigmentasyon kozmetik bireysel pazarlar için tercih cilt renkleri4karşılamak için geliştirilmiştir. Buna göre bilimsel ve pratik çalışmalar hipopigmentasyon ve anti-melanogenic aktivitesinin önemlidir.
Melanogenesis melanin biyosentezi melanosit vücudunda kimyasal reaksiyonlar enzimatik ve spontan bir dizi karmaşık işlemidir. Bir melanosit yaklaşık 36 lenfositi tarafından çevrilidir ve melanosit komşu lenfositi onların ürün dağıtmak melanin sentezini fabrikaları vardır. Deride, üretilen ve melanosit melanosomal bölümünde depolanan melanin dendrites epidermis ile komşu lenfositi için taşınır.
L-tirozin 3,4-dihydroxyphenylalanine (BASINCIN) ve sonra dopaquinone içine dönüştürmek iki ardışık tepkileri melanogenesis ve enzim Tirozinaz için ilk substrat tromboksan L-Tyrosine hizmet vermektedir. Bu tepkiler melanogenesis5,6hızı sınırlaması adım vardır. Buna göre hipopigmentasyon etkinlik ilk doğrudan hücresel Tirozinaz etkinliğini değerlendirmek tarafından ölçülebilir. Bunu yapmak için melanosit özleri içeren Tirozinaz BASINCIN ile inkübe ve örnekleri üretilen dopaquinone spectrophotometry, 475 tarafından ölçülebilir nm. Değerleri örnekleri ve hipopigmentasyon faaliyet sonucu denetimleri ile karşılaştırıldığında daha az dopaquinone oluşumunda maddelerle protein konsantrasyonları tarafından normalleştirilmiş.
İkinci olarak, hipopigmentasyon etkinlik melanin kültürlü melanosit içinde doğrudan ölçerek sayısal. Test materyalini hücrelerle tedavi sonra melanin alkali koşullarda elde edilir ve melanin içerik spectrophotometry 400 tarafından sayısal nm. Hipopigmentasyon Ajan daha düşük bir melanin içerik denetimleri7daha neden olur.
Son olarak, hipopigmentasyon etkinlik Fontana-Masson melanin boyama ve sonraki görüntü analizi tarafından sayısal. Fontana-Masson boyama, melanin granülleri amonyak-gümüş nitrat görünür bir siyah metalik durumuna azaltmak ve hücrelerin mikroskobik görüntüler siyah alanlar melanin miktarı gösterir.
Hipopigmentasyon Aracısı genellikle hangi madde etkinliği geçerli olduğunu doğrular Bu üç yöntem ile tutarlı ve benzer sonuçlar verir. Alternatif olarak, bu ifade anahtar genler ve proteinler arasında melanogenesis yanıt hipopigmentasyon etkinliğini incelemek için bir test madde olarak ölçmek yararlı olabilir. Tirozinaz yanı sıra, Tirozinaz kaynaklı proteinler (TRP-1) ve dopachrome tautomerase (TRP-2) kritik melanogenesis8enzimlerdir. Transkripsiyon faktörü mikroftalmia ilişkili transkripsiyon faktörü (MITF) melanogenesis ve miktar onun gen/protein ifade düzeylerinin ana bir düzenleyicisidir veya bir organizatörü etkinliği tahlil hipopigmentasyon etkinliğini değerlendirmek için de kullanılabilir.

<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

Bu çalışmada miktar hipopigmentasyon faaliyet tüp bebek için laboratuvar yöntemleri sunar. Melanin, melanosit, büyük pigment birden çok hücresel ve çevresel faktörler cevaben sentezlenir. Melanin cilt hücrelerini ultraviyole zararlardan korur, ama aynı zamanda biyofiziksel ve biyokimyasal işlevleri vardır. Aşırı üretimi veya melanosit melanin birikimi çiller, siyah noktaları, melazma ve benler gibi dermatolojik sorunlara neden olabilir. Bu nedenle, hipopigmentasyon ajanlar ile melanogenesis kontrolünü klinik veya kozmetik ihtiyaçları olan bireylerde önemlidir. Melanin öncelikle melanosit etkilenir melanogenesis dışsal olarak adlandırılan karmaşık biyokimyasal bir süreç ve hormonlar, iltihap, yaş ve ultraviyole ışık pozlama gibi Intrinsic faktörler melanosomes sentezlenir. Biz, kimyasal madde veya melanosit doğal maddelerin hipopigmentasyon etkinliğini belirlemek için üç yöntem tarif: 1) hücresel Tirozinaz etkinlik ve 2) melanin içerik ve görüntü ile 3) Boyama ve sayısal hücresel melanin ölçüm analiz.
Melanogenesis, L-tirozin 3,4-dihydroxyphenylalanine (L-DOPA) ve sonra dopaquinone içine dönüştürür hızı sınırlaması adım Tirozinaz tromboksan. Bu nedenle, Tirozinaz inhibisyonu bir birincil hipopigmentasyon mekanizmadır. Kültürlü melanosit içinde Tirozinaz etkinlik L-DOPA bir substrat ekleyerek ve dopaquinone üretim spectrophotometry tarafından ölçme sayısal. Melanogenesis da melanin içerik miktarının ölçmek. Melanin içeren hücresel kesir NaOH ile elde edilir ve melanin spectrophotometrically sayılabilir. Son olarak, melanin içerik Fontana-Masson melanin boyama aşağıdaki görüntü analizi tarafından sayılabilir. Her ne kadar bu tüp bebek deneyleri sonuçlarını insan cilt her zaman yeniden değil, bu yöntemler melanogenesis araştırma, potansiyel hipopigmentasyon etkinliği tanımlamak için özellikle ilk adımı olarak yaygın olarak kullanılmaktadır. Bu yöntemler, melanosit etkinlik, büyüme ve farklılaşma değerlendirmek için de kullanılabilir. Üç farklı yöntem tutarlı sonuçlar etkileri geçerliliğini sağlamak.

Arbutin, bir anti-melanogenic temsilcisi sonuçları hipopigmentasyon etkinliği için melanosit aşağıda gösterilmektedir B16F10 bileşik. Şekil 1A o arbutin, önemli ölçüde araç tedavi denetimi ile karşılaştırıldığında hücresel Tirozinaz etkinlik bastırılmış gösterir. Benzer şekilde, hücre arbutin ile uyarılan melanin içerik önemli ölçüde denetimleri (şekil 1B) ile karşılaştırıldığında dü...

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

Quantification of Hypopigmentation Activity In Vitro

Biz kimyasallar tüp bebek hipopigmentasyon etkinliğini değerlendirmek için üç deneysel yöntemleri tarif: miktar 1) hücresel Tirozinaz etkinlik ve 2) melanin içerik ve 3) melanin hücresel melanin boyama ve görüntü analizi tarafından ölçümü.

Miktar hipopigmentasyon aktivitesinin in Vitro

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

Bu yöntem, anti-melanojenik faaliyetleri ile fonksiyonel kozmetik ve cilt ajan gelişiminde yararlı olabilir. Bu gerçekleştirmek için var ve sonuç hızlı bir şekilde elde edilebilir. Buna ek olarak, bu yöntem etkileri veya bileşikleri tanımlamak veya araştırmak veya antimelanogenic veya promelanojenik faaliyetleri araştırmak isteyen araştırmacılar için yararlı olabilir.Tyrosinaz aktivitesi ölçmek için b16-F10 Melanicides beş kez 10 bir hücre kültürü kuluçka için tam ortamda 24 iyi plaka konsantrasyonu kuyu başına dördüncü hücrelere tohumlama ile başlar. Ertesi gün, yeni hazırlanmış test bileşiği ve inhibitör kontrolü veya negatif kontrol ile desteklenen DMEM ile her kuyuda supernatant değiştirin ve başka bir 72 saat için kuvöz plaka dönmek. Tedavi nin sonunda kuyuları kuyu başına 300 mikrolitre soğuk pb ile iki kez durulayın ve plakayı buza yerleştirin.Sonraki beş dakika sonra hücre lysates toplamak için bir hücre kazıyıcı kullanarak her iyi lysis tampon 300 mikrolitre ekleyin. Ortaya çıkan hücre parçacık süspansiyonlarını tek tek 1,5 mililitrelik mikro santrifüj tüplerine aktarın. Ve lisatları 14,500 rpm'de iki saniye boyunca üç kez homojenize edin ve hücreler arası Tyrosinaz'ı serbest bırakın.Santrifüj ile hücre enkaz pelet ve buz üzerinde bireysel 1.5 mililitrelik santrifüj tüpler için supernatants aktarın. Her bir süpernatantın 70 mikrolitresini 96 kuyunun tek tek kuyularına ayırın ve her bir süpernatant kuyuya 140 mikrolitre Tyrosinase substrat çözeltisi ekleyin. Iyi içeriğini karıştırmak için plaka nazik sallayarak.Sonra plakayı 37 derecede iki saat kuluçkaya yatırın. Ve bir mikroplaka okuyucu üzerinde 475 nanometre de Tyrosinaz aktivitesi ölçmek. Tedavi edilen hücrelerin lysate'sini topladıktan sonra hücrelerin melanin içeriğini ölçmek için.Santrifüj ile lysates toplamak ve yeni tüpler için supernatants aktarın. 60 santigrat derecede bir saatlik kuluçka için her pelet için normal bir sodyum hidroksit 300 mikrolitre ekleyin. Ardından çözünmüş hücre süspansiyonlarının santrifüjü.Daha sonra 200 mikrolitre süpernatantı 96 kuyunun her kuyuya aktarın. Ve 400 nanometre dalga boyunda bir mikroplaka okuyucu üzerinde melanin içeriğini ölçmek. Fontana-Masson Boyama için, tedavi edilen hücreleri 300 mikrolitre soğuk pbs ile iki kez yıkayın ve hücreleri 4 santigrat derecede bir saat boyunca %10 formalin 200 mikrolitre ile düzeltin.Kalın hücreleri distile suyla durulayın ve her kuyuya 58 ila 68 santigrat derece Amonyak gümüşçalışma çözeltisi 200 mikrolitre ekleyin. 37 santigrat derecede bir saat kuluçka ya da hücreler sarı-kahverengi renge dönüşene kadar. Lekeli hücreleri distile suyla durulayın ve ardından oda sıcaklığında %0,1 altın klorür içinde iki dakikalık kuluçka makinesi.Altın klorür işlenmiş hücreleri distile suyla durulayın ve hücrelere 200 mikrolitre sodyum tiyosülfat çözeltisi ekleyin. İki dakika sonra hücreleri tekrar durulayın. Ve hücreleri beş dakika boyunca her kuyuda 200 mikrolitre nükleer hızlı kırmızı ile etiketleyin.Daha sonra bir ışık mikroskobu altında görüntüleme önce musluk suyu ile lekeli hücreleri yıkayın. Eşik çözümlemesi için, Resim J'deki görüntüleri açın ve görüntüyü seçin, ayarlayın ve Renk Eşiği'ni seçin. Fontana Masson ile boyanmış alanı ölçmek için parlaklık parametre çubuğunu sıfıra ayarlayın ve parlaklığı siyah veya kahverengi lekeli hücrelerin tümünün dahil edildiği noktaya ayarlayın.Parçacıkları Analiz Et ve Analiz Et'i seçin ve Parçacıkları Analiz Et penceresinde özet kutusunu işaretleyin ve sonuçların bir özetini almak için tamam'ı tıklatın ve verileri elektronik tabloya kopyalayıp yapıştırın. Arbutin tedavisi önemli ölçüde kontrol aracı tedavi hücreleri ile karşılaştırıldığında hücresel Tyrosinaz aktivitesini bastırır. Benzer şekilde Arbutin ile uyarılan hücrelerin melanin içeriği kontrollere göre önemli ölçüde azalır.Hücreler tedavi grupları arasında morfolojik olarak benzer olmasına rağmen, Arbutin kontrol tedavi hücreleri ile karşılaştırıldığında siyah pigment alanını azaltır. Bu yöntem, bileşik kitaplığımızı kullanarak tarama faaliyetleri için yararlıdır. Hızlı bir şekilde test edilmesi gereken yüzlerce örnek vardır.Ayrıca bu yöntem melanogenez içeren mekanizmayı araştırmak için de kullanılabilir. Biyoaktif adaylar bileşikler tespit edildikten sonra, biyolojik mekanizmalar veya ticari uygulamalar çalışma onların tanıklığı için.

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.

pH bağımlı aktivitenin tespiti<em&gt; Escherichia coli</em&gt; Chaperone HdeB<em&gt; İn Vitro</em&gt; ve<em&gt; İn Vivo</em

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

Biyokimya

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.

Miktar bolluk ve Escherichia coli olarak Transfer RNA'ların düzeyde şarj

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.

Eşleme metabolizma: doğrudan dokusunda laktat dehidrogenaz etkinliğini izleme

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-Isopentenyl transferaz aktivite tespit etmek için bir In Vitro tahlil

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.

Kalibrasyon-Alerjik Vitro miktar Protein Homo-Oligomerizasyonda ticari araçları ve ücretsiz, açık kaynak parlaklık analiz yazılımı kullanarak

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.

Drosophila Melanogaster'de Sitrat Synthase Aktivitesinin Kolorimetrik Tsöplini

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).

Caenorhabditis elegans 2-Arachidonoylglycerol Quantification için Bir Deuterated Standart Sentezi

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.

Multipleksed Co-İmmünopreyağış Kullanarak Protein Etkileşim Ağı Dinamiğinin Ölçülmesi

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.

Hücre ve Dokularda Koenzim A'nın Sayısallaştırılması

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

mtDNA'nın sentezi ve dağılımının yüksek verimli miktar tayini

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.

Yazar Spotlight: Mitokondriyal DNA Sentezi ve Dağılımının Yüksek Verimli Görüntü Tabanlı Miktar Tayini  

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.

Mitokondriyal DNA Sentezi ve Dağılımının Yüksek Verimli Görüntü Tabanlı Nicelleştirilmesi

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