Bu çalışma, Ulusal Sağlık Enstitüsü (CA142664) tarafından desteklenmiştir.

1. Karboksi-H 2 DCFDA kullanarak ROS Algılama Karboksi-H 2 DCFDA floresan olmayan, fakat bu reaktif okside ROS varlığı, yeşil floresan olur. <ol><li> Hemen önce, steril dimethylsülfoksit (DMSO) veya% 100 etanol karboksi-H 2 DCFDA taze bir stok solüsyon hazırlanır. Boya birden fazla erime / donma döngüleri kaçının. </li><li> Hücrelerin Hepes ile yıkayın tamponlu tuz solüsyonu (HBSS) ya da salin (PBS) orijinal orta izlerini silmek için fosfat tamponlu. </li><li> Boya hücreleri (ve kontrol boya, cf not bölümünde) yükleyin. Bu testte biz Jurkat, bir insan lösemi hücre hattı kullanılmıştır. Azaltılmış serum (% 2) ile 1 mikrona kadar düzenli kültür ortamı bir final konsantrasyonda karboksi-H 2 DCFDA kullanın. </li><li> Geleneksel bir inkübatör (37 ° C,% 5 CO2) kültürlerin, karanlıkta 30 dakika inkübe edin. Kullanılmayan tüm boya çözümleri atın. </li><li> Karboksi çıkarın-H 2 DCFDA orta içeren ve HBSS veya PBS ile iki kez yıkayın. Bu adım ileri, ışık hücreleri korumak. </li><li> Karboksi-H 2 DCFDA yüklü hücreler için tercih edilen ilaç içeren taze orta ekleyin ve istediğiniz gibi inkübe edin. Bu örnek için H 2 O 2 (0.03%) 1 saat süreyle kullanın. </li><li> ROS hücrelerin hemen FL1 kanalı (yeşil floresans) ya da floresan plaka okuyucu tarafından veya floresan mikroskobu kullanılarak flow sitometri ile analiz ederek değerlendirin. Floresan yeşil floresan için uygun uyarma ve emisyon dalga boyları kullanarak tespit edilebilir. </li></ol> Not: Kontroller karboksi-H 2 DCFDA yüklenmiş tedavi edilmezse hücreleri ve lekesiz tedavi edilmezse hücreleri içermelidir. Karboksi-H 2 DCFDA peroksitler tespit bilinen değil, aynı zamanda diğer ROS tarafından okside olabilir. Bu reaktif da başka yollarla, örneğin 5 olarak oksidasyon duyarsız kontrolü boya değiştirilmiş olabilir (ve-6)-karboksi-2 &#39;, 7&#39;-dichlorBu nedenle test ofluorescein diasetat (karboksi-DCFDA) dahil edilmelidir. 2. Nitrik oksit (NO) üretimi ölçümü Sülfanilamid ve N-1-napthylethylenediamine dihidroklorür (NED) çözümleri ve Nitrit standart gerekecektir. Bu testte Griess analiz denir. NED çözüm:% 5 fosforik asit seyreltilmiş sülfanilamid% 1&#39;lik bir çözüm olun: N-1-napthylethylenediamine dihidroklorür steril water.Sulfanilamide çözelti içinde seyreltilmiş% 0,1 çözüm olun. Nitrit Standart: 100μM 0.1M standart stok sodyum nitrit sulandırınız steril ortamda, aynı ortamda bir seri seyreltme yapmak. Saklama Koşulları: oda sıcaklığında üreticisi tarafından yönetilen Mağaza kimyasallar. NED ve sülfanilamid çözümleri sulandırılmış zaman, karanlık, en fazla 3 ay, 4 ° C hemen sonra saklanır. <ol><li> 96 plaka Kültür hücreleri,kullanımı, her bir durum için triplicates ve deneylere göre uygun kontrolleri içermektedir. </li><li> NO üretimini neden hücreleri davranın. Deneyde bizim hücrelerin tedavisinde lipopolisakkarit (LPS) (100 ng / ml) ve rekombinant IL-4 kullanın. Bu protokol, RAW 264,7, bir fare makrofaj hücre hattı (Fare lösemiye monosit makrofaj hücre hattı) kullanılır. </li><li> Testin yapıldığı gün, hem reaktifler oda sıcaklığına getirin. </li><li> Plaka Spin hücre Süpernatantları toplamak ve yeni bir 96 plaka 50μl transferi. Bir standart eğri yapmak için standart stok solüsyonu seyreltme kuyu sınırlayıcı hazırlayın. </li><li> Her bir örnek ve iyi kontrol sülfanilamid Çözüm 50μl ekleyin ve iyice karıştırın. </li><li> Karanlıkta 10 dakika oda sıcaklığında inkübe edin. </li><li> , Her bir örnek ve iyi kontrol için N-1-napthylethylenediamine dihidroklorür çözüm 50μl ekleyin ve iyice karıştırın. </li><li> Karanlıkta 10 dakika oda sıcaklığında inkübe edin. </li><li> Absorba ölçün520Nm ve 550nm arasındaki dalga boylarında filtresi ile hemen bir plaka okuyucu kullanarak nce. </li></ol> Farklı plaka / çanak boyutları kullanmaktadır 1/1/1 hacmi, her çözümü ve örnek süpernatant için kullanın. Bir mor renk pozitif kuyularda görünecektir. Standardı ile elde edilen sonuçlar, çözümler istikrar kontrol yardımcı olacaktır. 3. ROS aksiyon Tespiti: okside proteinler ROS üretimi tanımlamak için farklı bir yöntem, proteinlerin oksidasyonu tespit ederek sonunda sonuçlarına bakmak için. Gerçekten de, ROS değiştirme glutatyon, birçok hücreleri dile getirdi ve bir antioksidan, ROS karşı koruyucu bir rol oynar. ROS bir disülfit köprüsü üzerinden ikinci bir glutatyon, sistein sülfidril (tiyol) grubu indirgenmiş glutatyon (GSH) sonuçları değişiklik ile oksidasyon ardından. (Okside protein GSSG) bir dimerized proteinin oluşumuna yol açar. GSH enzim glutatyon redüktaz tarafından GSSG değişiklik ile restore edilebilir. GSSG / GSH oranında artış oksidatif stres yansıtır. Aşağıdaki testi bu okside proteinlerin tespiti ve ölçülmesi dayanmaktadır. Bu yöntem, özel ROS için seçici değildir ama NO etkileri oldukça algılar, H 2 O 2, O 2 ve diğer ROS. Burada bioluminescent sinyalleri kullanarak okside (GSSG) ve düşük (GSH), glutatyon toplam miktarını ölçmek. <ol><li> Her zamanki gibi 96 plaka (şeffaf / beyaz, düz dipleri) Tohum hücreleri. Deneme için, biz yapışık Ham 264,7 ve A549 hücreleri ve süspansiyon Jurkat hücreleri için de ortalama 1x10 4 hücreler kullanıldı. Bu sayılar hücrelerinin büyüklüğüne göre ayarlanabilir. Test plaka takmak için izin önce plaka yapışık hücrelere gün öneriyoruz. Yeter kuyu gibi &quot;orta sadece&quot; için okside ve düşük protein tespiti için hazır olmalıdırkontrol grubu olarak tedavi edilmezse hücreleri. Kullanım koşulları için triplicates. </li><li> Gerekli zaman için ilaç hücreleri davranın. Burada H 2 O 2 (sırasıyla 5mM ve 2.5mm) ile Jurkat hücreleri ve A549 hücreleri için 1h davrandı. Ham 264,7 hücreler 16 saat boyunca LPS 200ng/ml ile tedavi edildi Bu inkübasyon süresi boyunca, tüm reaktifler oda sıcaklığında (RT) getirin. En fazla 30 dakika Testi gerçekleştirmeden önce tüm reaktifler olun. Aşağıdaki tablo, iyi ortalama reaktiflerin birimleri gösterir. Mümkün olduğunda, bu test ile devam etmeden önce indirgen içeren ortamı çıkarmak için önemlidir. </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> Yapışık hücrelere </td><td colspan="2"> Süspansiyon hücreleri </td></tr><tr><td> Toplam Glu Lizis </td><td> Okside Glu Lizis </td><td> Toplam Glu Lizis </td><td> Okside Glu Lizis </s&gt; Trong </td></tr><tr><td> Milli Ekonomi Modeli&#39;nde, 25mm </td><td> hiçbiri </td><td> 0.5μl </td><td> hiçbiri </td><td> 0.5μl </td></tr><tr><td> Luciferin-NT </td><td> 1 ul </td><td> 1 ul </td><td> 1 ul </td><td> 1 ul </td></tr><tr><td> Pasif Lizis Tampon, 5X </td><td> 10μl </td><td> 10μl </td><td> 10μl </td><td> 10μl </td></tr><tr><td> Damıtılmış su </td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> Final de ortalama hacmi </td><td> 50μl </td><td> 50μl </td><td> 25μl </td><td> 25μl </td></tr></tbody></table><ol start="3"><li> Yapışık hücreleri ile kuyulardan orta çıkarın. Süspansiyon hücreleri ile kuyu orta çıkarmayın. </li><li> Indirgenmiş glutatyon reaktifi veya okside glutatyon reaktifi ile ilgili kuyu ekleyin ve 5 dakika RT çalkalanır. </li><li> Luciferase nesil reaktif ve incubat eklee az 30 dakika RT. </li><li> Luciferase algılama reaktif ekleyin ve oda sıcaklığında 15 dakika inkübe edin. </li><li> 0,25-1 saniyede bir plaka okuyucu luminometre kullanarak entegrasyonu zamanda bioluminescent sinyal okuyun. </li></ol><table border="1"><tbody><tr><td></td><td> Yapışık hücrelere </td><td> Süspansiyon hücreleri </td></tr><tr><td> De ortalama hücre sayısı </td><td> 1x10 4 </td><td> 1x10 4 </td></tr><tr><td> Iyi + ilaç başına hücre süspansiyonu </td><td> 100 ul, 3.4 önce çıkartılmalıdır </td><td> 25μl, kaldırılacak değil </td></tr><tr><td> Glutathione reaktifi </td><td> 50 ul </td><td> 25μl </td></tr><tr><td> Okside glutatyon Lizis Reaktif </td><td> 50 ul </td><td> 25μl </td></tr><tr><td> Luciferase Üretimi Reaktif </td><td> 50 ul </td><td> 50 ul </td></tr><tr><td> Luciferase Algılama Reaktif </td><td> 100 ul </td><td> 100 ul </td></tr></tbody></table> 4. Temsilcisi Sonuçlar: <img alt="Şekil 1" src="/files/ftp_upload/3357/3357fig1.jpg" /> Şekil 1, karboksi-H2DCFDA boya kullanarak ROS Algılama . H 2 O 2 ile tedavi edilen Jurkat hücreleri (insan lösemi hücre hattı) ile tedavi olmayan hücrelere karşılaştırıldı . ROS flow sitometri, H 2 O 2 muamele edilen hücreler kontrolleri doruklarına (H 2 O 2 tedavili hücrelerde oksidasyon duyarsız boya ve sigara ile boyanmış kıyasla vardiya floresan pik tespit edilen yeşil fluoresces karboksi-H 2 DCFDA değişiklik neden olur karboksi-H 2 DCFDA ile boyanan ile tedavi edilen hücreleri). Sonuçlar, tedavi edilen hücrelerde ROS varlığını doğrulamaktadır. <img alt="Şekil 2" src="/files/ftp_upload/3357/3357fig2.jpg" /> Şekil 2 Algılama of Griess reaktifleri kullanarak YOK. RAW 264,7 hücreleri (fare makrofaj) LPS ve IL-4 ile tedavi edildi. Tedavi edilmezse hücreleri kontrol ile karşılaştırıldığında tedavi edilen hücrelerde NO üretiminin önemli bir artış tespit edildi. <table border="1"><tbody><tr><td> Hücre dizileri </td><td> Sigara ile tedavi edilen </td><td> Arıtılmış </td></tr><tr><td> Ham 264,7 </td><td> 13,0 </td><td> 8,3 </td></tr><tr><td> A549 </td><td> 21,6 </td><td> 10,5 </td></tr><tr><td> Jurkat </td><td> 5,2 </td><td> 2,8 </td></tr></tbody></table> Tablo 1 Algılama ROS aracılı protein oksidasyonu. RAW 264,7 LPS, Jurkat ve A549 hücreleri ile tedavi edildi (insan akciğer kanserli) hücreler H 2 O 2 ile tedavi edildi. Sonuçlar oranı azalır (GSH) / (GSSG) glutatyon okside olarak ifade edilir. Glutatyon (GSH) / (GSSG) daha düşük oranlara göre muamele edilen hücreler tespit edildiproteinler muamele edilen hücreler okside olduğunu açıklayan, işlenmemiş hücreleri kontrol eder.

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, 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=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

Biz, bu yayın için Promega destek aldı.

Inflamatuar hastalıklar, kanserler, iskemi / reperfüzyon, ve aynı zamanda, radyasyon veya kemoterapi gibi tedaviler gibi bazı patolojik durumlarda (yani sisplatin) ROS aşırı üretimi neden olur. Böylece, tespit etmek ve ROS düzeylerini ölçerek çok basit, klinik öncesi ve klinik çalışmalar önemlidir. Ancak, ROS çok kısa yarı ömre sahip ve tespit etmek için karmaşık olabilir. Burada, memeli hücreleri serbest radikal üretiminin tespiti için rutin olarak kullanılan ve yaygın olarak kabul edilmek...

<table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> 5 - (6)-karboksi-2 &#39;, 7&#39;-dichlorofluorescein diasetat (karboksi-DCFDA) </td><td> Moleküler Probları </td><td> C369 </td><td> kontrol </td></tr><tr><td> karboksi-H 2 DCFDA </td><td> Moleküler Probları </td><td> C400 </td><td></td></tr><tr><td> Sülfanilamid </td><td> Sigma </td><td> S9251-100G </td><td></td></tr><tr><td> N-1-napthylethylenediamine dihidroklorür </td><td> Sigma </td><td> N9125-10G </td><td></td></tr><tr><td> Nitrit standart </td><td> Sigma </td><td> 237.213-100G </td><td></td></tr><tr><td> GSH / GSSG-Glo Testi </td><td> Promega </td><td> V6612 </td><td> Ölçmek, okside azaltılabilir veya okside / glutatyon azaltılmış </td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

Reaktif oksijen türleri, moleküllerin bir dizi zarar veren DNA ve RNA ve proteinler ve lipitler (lipit peroxydation) okside. Bu reaktif moleküller oksijen içeren ve H 2 O 2 (hidrojen peroksit), NO (nitrik oksit), O 2 - (oksit anyon), peroksinitrit (ONOO -), hydrochlorous asit (HOCl) ve hidroksil radikal (OH -) . Oksidatif türlerin sadece patolojik durumlarda (kanser, iskemik reperfüzyon /, nörolojik ve kardiyovasküler patolojiler, enfeksiyon hastalıkları, inflamatuar hastalıklar 1, otoimmün hastalıklar 2, vb ...) altında değil, aynı zamanda bu tür hücre metabolizması 3 gibi fizyolojik (non-patolojik) durumlarında üretilir , 4. Nitekim, ROS (çoğalması, hücre aktivasyonu 5, 6, 7 göç vb.) Birçok hücresel sinyal yolları önemli rol oynamaktadır. ROS zararlı olabilir (o olarak adlandırılır, Hücre içi bölmeleri ve hücreler yüksek miktarda üretilen &quot;ve nitrosatif oksidatif stres&quot;) genellikle süperoksit dismutaz (SOD) ve katalaz (CAT), glutatyon peroksidaz (GPx) ve koruyan glutatyon (GSH) gibi antioksidanlar upregulating ROS yanıt tehlikeli serbest radikalleri zararsız moleküller (yani su) dönüştürerek. C ve E vitaminleri de ROS temizleyiciler (antioksidan) olarak tarif edilmiştir. Serbest radikaller, düşük miktarda 3 yararlıdır . Makrofaj ve nötrofil-aracılı bağışıklık yanıtlarını, virüsler, patojenler ve tümör proliferasyonu 8 inhibe NO üretimi ve salınımı, içerir . NO aynı zamanda diğer ROS ile reaksiyona girer ve böylece, aynı zamanda bir detoks (ROS çöpçü) olarak bir rolü vardır. Uzun süren egzersiz 9, 10 kas adaptasyon için önemli olan kan akışını düzenlemek için damarları üzerinde Sonunda YOK davranır. Çeşitli yayınlar da ROS insülin sens yer aldığını göstermiştiritivity 11, 12. ROS üretimini değerlendirmek için çok sayıda yöntem mevcuttur. Bu yazıda, birkaç basit, hızlı ve uygun fiyatlı testleri önerebilir; bu testlerin birçok yayın tarafından onaylanmıştır ve memeli hücrelerinde ROS veya onun etkilerini tespit etmek için rutin olarak kullanılmaktadır. Bu testlerin bazıları birden fazla ROS tespit ederken, diğerleri sadece tek bir ROS algılar.

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

Burada hücreler reaktif oksijen türlerinin varlığını test etmek ve değerlendirmek için basit yöntemler öneriyoruz.

Kanserler Üretim ve reaktif oksijen türlerinin (ROS Algılama)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.9K Görüntüleme.  Baylor College of Medicine. Burada hücreler reaktif oksijen türlerinin varlığını test etmek ve değerlendirmek için basit yöntemler öneriyoruz.

Video: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Akut Hepatit Model Uygulama: Görselleştirme ve Hepatik Mikrosirkülasyon Kan Akış ve Oksijen Tüketimi Analizi

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

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Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Kanser Tedavisi İzleme Cerenkov Lüminesans Görüntüleme (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Bir Biyoassay olarak Sığır Mezenterik Arter ve Ven Bölüm yalıtma ve kullanma İnce Barsak vazoaktiviteye için Test

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Bir ile Bulaşıcı Süngerimsi Ensefalopati Türler Engelleri değerlendirilmesi<em&gt; İn Vitro</em&gt; Prion Proteini Dönüştürme Deneyi

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

İnsan A53T aşırı eksprese eden transjenik farelerin beyninde Geliştirilmiş ELISA ile Hastalık ile ilişkili α-sinüklein tespiti α-sinüklein Mutasyona uğramış

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Reaktif gliozis Tetikleme<em&gt; In Vivo</em&gt; Bir Önbeyin Stab Yaralanma tarafından

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Nitrik oksit VASP fosforilasyon ölçme tarafından kan trombosit tabanlı algılama

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Proton manyetik rezonans görüntüleme ve oksijen kontrast ajan olarak kullanarak ınsan akciğerinde spesifik havalandırma niceliksel haritalama

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

Oksijen Gradyan Ektasittometri ile Kontrollü Otomatik Deoksijenasyon Sırasında Hastalık Karakterizasyonu

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...