Bu çalışma NIH Ulusal Kalp, Akciğer ve Kan Enstitüsü hibe RO1 HL81248 tarafından finanse edildi.

1. Sığır aort endotel hücreleri (BAEC) Kültür <ol><li> Uygun aseptik teknikler takip edildi. </li><li> 37 antibiyotik olmayan bir su banyosu, sıcak ortamda ° C. </li></ol> Not: orta 4.5 g / L D-glikoz ile fenol serbest Dulbecco&#39;nun değiştirilmiş Eagle ortamı (DMEM) oluşur,% 10 fetal bovin serumu (FBS) ve 2.5 ile desteklenmiş 4 mM L-glutamin,% 1 olmayan amino asitler, mg / L endotelyal büyüme faktörü. <ol start="3"><li> Inkübatör gelen T75 şişeyi içeren hücrelerin çıkarın ve kapağı içine yerleştirmeden önce% 70 etanol ile balonun yüzeyi temizleyin. </li><li> Bir aspiratör kullanarak eski orta çıkarın ve Dulbecco fosfat tamponu salin (DPBS) 5 ml iki kez yıkayın. </li><li> Tripsin 2 ml ekleyin ve periyodik mikroskop altında incelerken ayırmak için hücreler için 4-5 dakika bekleyin. </li><li> Orta 3 ml ekleyin ve tekrar tekrar ayrı t için bir pipet ile karıştırıno hücreleri ve hatta süspansiyon oluşturun. </li><li> 5 dakika boyunca 121 g bir 15 ml tüpü ve santrifüj ile tripsin ile orta 5 ml transfer edin. </li><li> Bir aspiratörü kullanarak süpernatantı. DPBS 5 ml ekleyin ve iyice karıştırın. 5 dakika 121g santrifüjleyin. </li><li> Süpernatant aspire ve orta 6 ml ekleyerek hücre peleti tekrar askıya. </li><li> Bir 6-kuyucuklu plak üzerinde, çok daha sonra, orta ve 1 ml ilave her bir hücre süspansiyonu 1 ml ilave edilir. Bir pipet kullanarak süspansiyonu karıştırın. </li><li> Plakası etiketlemek ve 37, bir gece boyunca inkübe ° C&#39;de ve% 5 CO2. </li></ol> 2A. BAEC Hücre ile NO Algılama <ol><li> Uygun aseptik teknikler takip edildi. </li><li> Kuluçka den 6-kuyucuklu plak kaldırmak ve birinci ve ikinci orta aspire. 1 ml DPBS ile iki kez yıkanır hücreleri. </li><li> 1.9 mM demir (II) sülfat heptahidrat (FeSO 4 .7 H 2 O, 0,8 mg eriterek taze hazırlamak 210 ul ekle01:07 oranında kullanılarak 1 CaCI2 ve MgCl2 ile DPBS ml) ve amonyum N-metil-D-glukamin ditiokarbamat (MGD, CaCI2 ve MgCl2 içeren 500 ul DPBS içinde 2.7 mg çözülerek taze olarak hazırlamak) arasında 210 ul . (Not:. aşırı MGD kullanılır Bu oran, geleneksel Fe 3 verim +-MGD aşırı MGD varlığında maksimize olmasından dolayı +-MGD kompleksi Fe 2 hazırlanması için kullanılan edilmiş bir ek Fe 2 stabilize etmek için çözelti içinde askorbat +-MGD NO-Fe 3 yana oluşan +-MGD endojen askorbat, hidrokinon, ya kadar olan dönüşüm verimle sistein ile +-mgd 2 NO-Fe EPR saptanabilir indirgenir gerekli değildir % 99,9 &#39;.. Fe 2 düşük spin diamanyetik devlet + NO) düşük sıcaklık cihazın gerek kalmadan düz hücre veya kapiler tüpü kullanarak 15 algılanmasını sağlar. </li><li> Swirl edilen süspansiyon iyi ve 4.6 μ ekleyinkalsiyum iyonofor l (CAI) (1 ml DMSO içinde 1 mg eriterek 1,9 mM bir stok çözeltisi hazırlanır.) </li><li> Swirl yine çözüm ve sırayla 36 dakika ° C daha-Fe 3 NO azaltılması NO-Fe 2 +-MGD EPR tespit etmek +-MGD izin vermek için 37 de inkübe edilir. </li><li> Bir Eppendorf tüp ve EPR yassı hücreli (veya 50 ul kılcal tüp) için transfer süpernatant (425 ul) toplayın. </li><li> EPR satın parametreler şunlardır: mikrodalga frekansı: 9.8 GHz; merkezi alanı: 3427 G, modülasyon genliği: 6.0 G; süpürme genişliği: 100 G; alıcı kazancı: 1 x 10 5; mikrodalga gücü: 10 mW; taramaları toplam sayısı: 121; tarama süresi: 10 sn; ve zaman sabiti: 20 ms (Not: parametreleri tek enstrüman ve deneysel koşullar diğerine değişir bu yana, bu nedenle, sadece merkez alan, frekans ve modülasyon genliği dikkat edilmesi gereken en önemli parametrelerdir.). </li><li> Oda sıcaklığında spektrumları kaydetme ve 2-D spektrumları INTEG edildiBruker WinEPR Bilgi İşlem yazılım veya diğer veri işleme yazılımı kullanılarak düzeltilebilir arka plan gürültü ve bazal azaltmak için puan. Adduct oluşumu ölçümü için, konsantrasyon karşı sinyal yoğunluğu (veya bölge) standart araziler bir donör SNAP NO kullanarak inşa edilebilir. </li></ol> 2B. eNOS ayrılmasının Deney <ol><li> Kuluçka den 6-kuyucuklu plak kaldırmak ve ikinci kuyudan orta aspire ve 1 ml DPBS ile iki kez yıkanır. </li><li> A peroksinitrit donör, 5-amino-3-(4-morfolinil) -1,2,3-oxadiazolium klorür (SIN-1) eNOS 16 çözüme kavuşturmak için kullanıldı. 0,5 mM SIN-1 (M r 206,6 taze olarak herhangi bir Ca / Mg iyonları ile 500 ul PBS içerisinde 1 mg sin-1 çözülmesiyle hazırlanmıştır 10 mM stok çözeltisinden g / mol) ve seyreltik ila 2 ml ile DPBS 100 ul ekleme ve% 10 FBS. </li><li> 37 de 2 saat süre ile inkübe ° C&#39;de ve% 5 CO2. </li><li> Inkübatör gelen plaka çıkarın ve DPBS iki kez yıkayın. </li><li> 210 ul ekle2,8 mM FeSO 4 0,7 H2O ve 19.6 mM 210 ul taze yukarıda belirtilen yönteme göre hazırlanan mgd. </li><li> Swirl çözüm ve 1.9 mM Cai 4.6 ul ekleyin. </li><li> Swirl yine çözüm ve 36 dakika 37 ° C&#39;de inkübe edin. </li><li> Bir Eppendorf tüp ve EPR düz hücre transferi (425 ul) süpernatant toplayın. </li><li> EPR satın parametreler şunlardır: mikrodalga frekansı: 9.8 GHz; merkezi alanı: 3427 G, modülasyon genliği: 6 G; süpürme genişliği: 100 G; alıcı kazancı: 1 x 10 5; mikrodalga gücü: 10 mW; taramaları toplam sayısı: 121; tarama süresi: 10 sn; ve sabit süresi: 20 ms. </li><li> Spektrumları kaydedin ve 2-B spektrumları yukarıda belirtildiği gibi düzeltilmiş arka plan gürültü ve bazal azaltmak için entegre edildi. </li></ol> 3. O 2 tespiti • - polimorfonükleer nötrofiller den (PMN) <ol><li> Daha önce tarif edildiği gibi 17, insan nötrofil kan örneği ile izole edildi. </li><li> Dietilentriamin-pentaasetik asit (DTPA) 0.1 mM içeren PBS içinde 1 M * DMPO bir stok çözelti yapmak. DMPO 25-29 bir erime noktasına sahiptir ° C de bir cam şişeye (Not sıvı DMPO (yoğunluk ~ 1.02 g / ml 25 ° C) Pipeti daha kullanışlı hale getirir: saf DMPO beri tartım için plastik şişeleri kullanmayın ) plastik ile reaksiyona girer. Dondurulmuş DMPO flakon (: DMPO bozulabilir olarak sıcak su tüketmemek Not) üzerine ılık su çalıştırarak eritebilir. </li><li> O zamandan beri yüksek saflıkta DMPO (&gt;% 99) kullanılması önemlidir ticari olarak mevcut sıkma tuzakları bazı paramanyetik safsızlıklar içerir, ve bu yüzden, sadece tek başına DMPO çözeltisi EPR tayfı (bu durumda, 10 mM) çalıştırmak için zaruridir. Bu arka plan sinyali (Şekil 3A bakınız) bellidir önemlidir. </li><li> DMSO içinde forbol-12-miristat-13-asetat (PMA) bir stok çözelti (1 mg / ml) hazırlamak. 10 mikrogram / ml PBS içinde çözüm seyreltilmesiyle alikotları olun. </li><li> Bir 1 &#39;de.5 ml Eppendorf tüpüne, ek olarak, sırası takip ederek 0,6 ml&#39;lik toplam hacmi, bir solüsyon hazırlanır: PMN ml başına 10 ~ 6 hücreleri, D-glükoz (1 mg / ml) ve albumin (1 mg / ml), 10 mM DMPO ve PMA ile 0,2 mg / ml. (Not: PMA radikal aktivatörü ve son olarak ilave edilmelidir). </li><li> EPR yassı hücreli çözüm aktarın. </li><li> EPR satın parametreleri Mikrodalga frekansı: 9.8 GHz; merkezi alanı: 3486 G, modülasyon genliği: 0.5 G; süpürme genişliği: 100 G; alıcı kazancı: 5 x 10 5; taramaları toplam sayısı: 10; tarama süresi: 30 s ; mikrodalga gücü: 20 mW; ve zaman sabiti: 81 ms. Adduct oluşumu ölçümü için, konsantrasyon karşı sinyal yoğunluğu (veya bölge) standart araziler gibi TEMPO veya 3-karboksilik asit-PROXYL olarak istikrarlı nitroxides kullanarak inşa edilebilir. </li></ol> DMPO kullanımına * önemli not: düz bir hücre kullanılarak, tek bir hücre yoğunluğu artırır ve böylece sinyali artan edebilirSpin adduct ama O 2 &#39;nin yarı ömrü yoğunluğu • - DMPO of adduct DMPO-OH parçalanmasına neden olan (t 1/2 ~ 1 dak) kısa. DMPO t ​​1/2 ~ 8 ve sırasıyla 14 dakika olan artan adükt stabilite için ticari olarak temin edilebilir EMPO, BMPO ya DEPMPO aynı konsantrasyonu kullanılarak ikame edilebilir. 4. Temsilcisi Sonuçlar Spin NO radikal yakalama Fe 2 +-MGD. Şekil 2A ve 2B ile yapıldı Fe 2 hiçbir EPR sinyal göstermiyor +-MGD veya Fe 2 karışımı arka plan sinyali YOK Bu reaktiflerin kaynaklı olduğunu gösteren, CAI ile +-MGD. CAI ile uyarılması üzerine BAEC Fe 2 ile reaksiyona girerek hangi NO bültenleri spin katkısı, NO-Fe 2 +-MGD oluşturmak için +-MGD, ve aşırı yarılma sabiti (hfsc) N = 12.66 G değeri ve karakteristik üçlü sinyali gösteriyor g = 2.040 g-faktörü. (Şekil 2C). Hfsc değeri NIEHS EPR Yazılım veritabanı web sitesinden indirilebilir WINSIM simülasyon programı kullanılarak belirlendi. Deneysel hfsc NO-Fe-2 + MGD adükt için bir N = 12,70 ve G = 2,041 g, 18 literatürde değeri ile tutarlıdır. Benzer şekilde, BAEC üzerine SIN-1 etkisi Şekil 2B&#39;de gösterildiği gibi ayrılmasının nedeni eNOS üretimi NO indirilir. Algılama - DMPO sıkma tuzağı O 2 • için kullanılmıştır. Spin tuzak paramanyetik pislik olduğunu onaylayan Şekil 3A gösterildiği gibi tek başına DMPO bir sinyal vermedi. Şekil 3B DMPO spektrumu ve PMN sadece ve benzer şekilde, DMPO neden olmaz düşündüren saptanabilir sinyal yok Enzim NADPH oksidaz aktivasyonu. Şekil 3C PMA ile PMN stimülasyonu üzerine gözlemlenen EPR sinyali gösterir. Bu sinyal için bir değer hfsc N = 14,71 G, bir &amp; olarak belirlenmiştirbeta;-H = 11,40 G ve bir γ-H = 1.25 g, ve bir N = 14.3 G, bir β-H = 11.7 G ve bir γ-H literatürde değerleri DMPO-O için = 1.3 g 19 ile tutarlıdır 2 H adükt. <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="Şekil 1" /> Şekil 1. EPR Spin hapsi kullanarak nötrofiller ve BAEC gelen radikallerin tespiti için akış çizelgesi. (A) PMNs&#39;nin DMPO ve PMA ile karıştırılmış ve ortaya çıkan karışım verilerini elde etme için bir düz EPR hücre aktarılır. (B) BAEC bir plaka üzerinde yetiştirilir ve DPBS ile yıkandı. Spin tuzak Fe (MGD) 2 CAI ile birlikte eklendi. Çözeltisi iyice karıştırılır ve inkübe edildi. Karışım EPR veri toplama için bir EPR düz hücreler nakledildi. <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="Şekil 2" /> B&#39;den NO Şekil 2. EPR tespitiAEC. Fe 2 (A) Spectrum +-MGD sadece (B) +-MGD + CAI sadece Fe 2 Spektrum. (C) Fe 2 NO hapsi kaynaklanan Üçüz spektrumu +-MGD kullanarak hücrelerin CAI-uyarılmış. (D) nedeniyle 0,5 mM hücrelerinin SIN-1 tedaviye NO üretimindeki düşüş gösteren Spectrum. <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="Şekil 3" /> Aktif hale gelen nötrofiller DMPO-O 2 H Şekil 3. EPR algılama. 10 mM DMPO sadece (A) Spektrumu. (B) 10 mM DMPO varlığında başına PMN Spektrumu. (C) PMN Spektrumu 10 mM DMPO varlığında PMA ile aktive.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+spin+trapping+properties+by+beta-cyclodextrin-cyclic+nitrone+conjugate.">Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate.</a> J. Org. Chem. 73, 7108-7117 (2008).</li><li>Hardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+characterization,+and+decay+kinetics+of+ROS+and+thiyl+adducts+of+mito-DEPMPO+spin+trap.">Detection, characterization, and decay kinetics of ROS and thiyl adducts of mito-DEPMPO spin trap.</a> Chem. Res. Toxicol. 20, 1053-1060 (2007).</li><li>Kim, S. -. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+reactivity+of+a+cyclic+nitrone-calix[4]pyrrole+conjugate+with+superoxide+radical+anion:+Theoretical+and+experimental+studies.">Fast reactivity of a cyclic nitrone-calix[4]pyrrole conjugate with superoxide radical anion: Theoretical and experimental studies.</a> J. Am. Chem. Soc. 132, 17157-17173 (2010).</li></ol>

Çıkar çatışması ilan etti.

EPR Spin hapsi serbest radikaller ve niceleme tanımlamak için biyomedikal uygulamalarda geniş bir istihdam edilmiştir. Sıkma bindirme derece hassas nM den böylece biyolojik sistemler uygulama için uygun hale uM arasında değişen konsantrasyonlarda radikalleri saptayabilir. Paramanyetik katkısı, NO-Fe 2 +-MGD, oluşumu EPR ile NO algılama temelidir. ~ Oranında 10 6 M -1 s -1 NO hızla 18 Fe 2 +-MGD tepki. NO-Fe 2 +-MGD addu...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Fenol ücretsiz DMEM Yüksek glukoz 1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> % 0.25 tripsin-EDTA </td><td> GIBCO </td><td> 25200 </td><td></td></tr><tr><td> L-Glutamine </td><td> Fisher Scientific </td><td> BP379-100 </td><td></td></tr><tr><td> MEM Olmayan Esansiyel Amino asitler </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Sığır fetüs serumu </td><td> Atlanta Biologicals </td><td> S11550 </td><td></td></tr><tr><td> Endotelyal büyüme faktörü </td><td> Millipore </td><td> 02-102 </td><td></td></tr><tr><td> Cai </td><td> Enzo Yaşam Bilimleri </td><td> A-23187 </td><td> DMSO içinde eritilir </td></tr><tr><td> SIN-1 </td><td> Enzo Yaşam Bilimleri </td><td> BML-CN245-0020 </td><td></td></tr><tr><td> DMPO </td><td> Dojindo Laboratuvarları </td><td> D048-10 </td><td></td></tr><tr><td> FeSO 4 .7 H 2 O </td><td> Sigma Aldrich </td><td> 215.422-250G </td><td> Ca ve Mg ile birlikte, PBS içerisinde çözünmesi </td></tr><tr><td> MGD </td><td> Enzo Yaşam Bilimleri </td><td> ALX-400-014-M050 </td><td> Ca 2 + ve Mg 2 ile PBS içinde eritilir + </td></tr><tr><td> BAEC hücreleri </td><td> Hücre Sistemleri </td><td> 2B2-C75 </td><td></td></tr><tr><td> DMSO </td><td> Fisher Scientific </td><td> BP231-100 </td><td></td></tr><tr><td> DPBS </td><td> Sigma Aldrich </td><td> D8537 </td><td></td></tr><tr><td> CaCI2 ve MgCl2 ile DPBS </td><td> Sigma Aldrich </td><td> D8662 </td><td></td></tr><tr><td> Forbol miristat asetat-(PMA) </td><td> Sigma Aldrich </td><td> 79.346-1MG </td><td></td></tr></tbody></table>

detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps

Düşük konsantrasyonlarda reaktif nitrojen / oksijen türlerinin (ROS / RNS) sinyalizasyon, hücre fonksiyonunu düzenlenmesinde önemli bir rol oynayabilir ve bağışıklık yanıtı ancak düzenlenmemiş konsantrasyonlarda hücre canlılığı 1, 2 için zararlıdır. Canlı sistemlerde ROS üretimi düzenleyen endojen ve diyet antioksidan savunma mekanizmaları ile büyürken, ROS normal oksijen metabolizmasının yan ürünleri doğal olarak sürekli olarak üretilir ve protein fonksiyon kaybı, DNA bölünme veya lipid sonuçlanan biyomoleküllere oksidatif hasara neden olabilir peroksidasyonu 3 ve sonuçta hücre hasarına ya da ölüme yol açan 4 oksidatif stres. Süperoksit radikal anyonu (O 2 • -) gibi peroksinitrit ve radikal hidroksil gibi biyolojik sistemler mevcut olduğu bilinmektedir en çok oksitleyici türler bazı önemli öncüsüdür. O 2 kuşak • - dolayısıyla oksidatif patlama ilk işareti işaret ve its algılama ve / veya biyolojik sistemler sekestrasyon önemlidir. Bu gösteri de, O 2 • - polimorfonükleer nötrofiller (PMN) elde edildi. Forbol-12-miristat-13-asetat (PMA) ile kemotaktik uyarılması yoluyla, PMN O 2 • üretir - nikotinamid adenin dinükleotid fosfat (NADPH) oksidaz 5 aktivasyonu yoluyla. Üç izoformları gelir nitrik oksit (NO) sentaz, indüklenebilen-, nöronal-ve endotel-NOS veya iNOS, nNOS ve eNOS, sırasıyla, NO 6 üretmek için, NADPH ile, L-sitrulin, L-arjininin dönüşüm katalize olarak . Burada, endotel hücrelerinden NO oluşturulur. Oksidatif stres koşulları altında, örneğin eNOS O 2 • NO üreten geçiş yapabilirsiniz - Heme 7 veya co-faktör, tetrahidrobiyopterin (BH 4) 8 oksidasyonu neden olduğuna inanılan ayrılmasının adı verilen bir süreç içinde. Sadece birkaç vardırancak biyolojik sistemlerde serbest radikallerin saptanması için güvenilir yöntemler özgüllük ve duyarlılığı ile sınırlıdır. Sıkma bindirme yaygın olarak serbest radikallerin belirlenmesi için kullanılan ve elektron paramanyetik rezonans (EPR) spektroskopi ile tespit edilebilir bir kalıcı sıkma adükt oluşturan bir spin tuzağı bir radikal ilavesiyle reaksiyon kapsamaktadır. Çeşitli radikal adducts oluşturulmakta radikalleri tespit etmek ve radikal üretiminin 9 doğasına ve kinetik hakkında bilgi zenginliği sağlayabilir kullanılabilir ayırt edici spektrumu sergilemesi. Siklik nitrones, 5,5-dimetil-pirolin-N-oksit, DMPO 10, fosforil-ikameli DEPMPO 11 ve ester-ikameli, EMPO 12 ve 13 BMPO, yaygın olarak dönme tuzağı olarak kullanılan edilmiştir - ikinci dönme adduct - tuzakları O 2 • için Uzun yarılanma ömrü sergileyerek. Demir (II)-N-metil-D-glukamin ditiokarbamat, Fe (MGD) 2 &lt;Alt /&gt; genel adükt oluşumu ve sıkma adükt 14 arasında yüksek stabilite yüksek oranlı NO bağlı olarak yakalamak için kullanılır.

Watch this Scientific Journal Video about Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps at JoVE.com

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Elektron paramanyetik rezonans (EPR) spektroskopisi demir (II)-N-metil-D-glukamin ditiokarbamat, Fe (MGD) kullanılarak, insan nötrofil gelen sığır aort endotelyal hücreler ve süperoksit anyon radikal nitrik oksit tespit etmek için kullanılmıştır<sub&gt; 2</sub&gt; Ve 5,5-dimetil-1-pyroroline-N-oksit, sırasıyla, DMPO,.

Nitrik Oksit ve hücreler arası Elektron Paramanyetik Rezonans Spektroskopisi ile süperoksit Radikal Anyon tespiti Spin Tuzakları kullanarak

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

detection-nitric-oxide-superoxide-radical-anion-electron-paramagnetic

Research

JoVE Journal

Biology

18.9K Görüntüleme.  The Ohio State University. Elektron paramanyetik rezonans (EPR) spektroskopisi demir (II)-N-metil-D-glukamin ditiokarbamat, Fe (MGD) kullanılarak, insan nötrofil gelen sığır aort endotelyal hücreler ve süperoksit anyon radikal nitrik oksit tespit etmek için kullanılmıştır 2 Ve 5,5-dimetil-1-pyroroline-N-oksit, sırasıyla, DMPO,.

Video: Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

Elektron Spin Rezonans Oksijen Haritalama için Canlı Türlerinin Micro-görüntüleme

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Biyoloji

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

C2C12 Myoblast Hücreler kullanarak Bioenerjetik bilgileri Deney

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Nükleer Manyetik Rezonans Spektroskopisi ile Çevre metabolomik için düşük yoğunluklu Planktonik topluluklardan Metabolitlerinin Konsantrasyon

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Live Algılama<em&gt; Escherichia coli</em&gt; O157: PMA-qPCR H7 Hücreler

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Hücre içi Ca İki foton Görüntüleme<sup&gt; 2+</sup&gt; Bir İzole Sıçan Aort Kullanım ve Endotel Nitrik Oksit Üretimi ve düz kas hücreleri

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Bozulmamış Arterlerin endotel katmanı Nitrik Oksit ve Süperoksit En Yüz Algılama

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Gelişim ve karakterizasyonu<em&gt; İn Vitro</em&gt; Mikrodamar Ağ ve Endotel kantitatif ölçümleri [Ca<sup&gt; 2+</sup&gt;]<sub&gt; I</sub&gt; Ve Nitrik Oksit Üretimi

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Hassas immünohistokimyasal sitozin Modifiye Formlar Algılama

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Kemilüminesans Yöntemi kullanılarak biyolojik Malzemeleri, Nitrik Oksit Yolu&#39;ndaki nitrit ve nitrat, Metabolitler Ölçme

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...