Bu çalışma NIH RO1 AI079253 ve Gazi İşleri Bakanlığı tarafından finanse edildi.

1. Hayvan Modelleri <ol><li> Fare: S47 phox-Kullanım / - farelerde, yaş ve cinsiyete göre eşleştirilmiş C57BL6/DBA fareler. Kurumsal Hayvan Bakım ve Kullanım Komitesi deneyler için onay alın. </li><li> Anestezi: Anestezi ikna etmek için sürekli bir izofluran yönetim sistemi kullanın. Buharlaştırıcı sistemi (VetEquip) (% 2-3) izofluran ile doldurulur. Fareler dış uyaranlara yanıt olarak solunum, hareket ve korneal refleks gözlemleyerek tamamen uyuşturulduktan olduğunu onaylayın. </li><li> Cerrahi yöntemler:% 70 etanol ile cerrahi alan (benchtop) fırçalayın. Steril eldiven ve temiz bir cerrahi önlük ve maske takın. Saç kırpma ve kesik olan alanı için% 70 etanol ile dönüşümlü olarak betadin uygulayarak fareler hazırlayın. Steril bir yüzey üzerinde ameliyat gerçekleştirin ve steril aletler kullanın. Monitör fareler postoperatif kadar uyanık ve serbestçe hareket. </li><li> Intratrakeal zymosan yönetimi <ol><li> Anesteziklereolarak 1.2 nitelendirdi. </li><li> Betadin ve% 70 etanol ile cerrahi alan (boyun) Dezenfekte ve cerrahi diseksiyon ile trakea maruz. </li><li> Pierce, 27-gauge iğne ile soluk borusu ve steril PBS içinde eritildi, 0.5 ug / ml &#39;den çözeltisi (25 g fare için toplam hacim 50 ul) ile 1 ug / gr&#39; lik bir dozda zymosan (St. Louis, MO) enjekte edilir. </li><li> Kapat fareler boyun aseptik koşullarda 5-0 steril ipek dikişlerle yara. </li><li> 4 saat ve 24 saat sonra resmi. </li></ol></li><li> Çekal ligasyon ve delme <ol><li> 1.2 &#39;de tanımlanan anesteziklere. </li><li> Ameliyat bölgesinin (karın) saç Klip ve betadin ve% 70 etanol ile dezenfekte edin. </li><li> Bir orta hat laparotomi gerçekleştirin ve çekum tanımlamak. </li><li> 4-0 ipek sütür ve 21-gauge iğne tek geçişe sahip ligasyonu distal ponksiyon çekum ile maruz çekum distal 50% Arter. </li><li> 4-0 steril ipek dikişlerle kesi kapatın. </li><li> 4 saat sonra görüntü ve24 saat. </li></ol></li><li> Sözlü karbon tetraklorür (CCl4) idaresi <ol><li> 1.2 &#39;de tanımlanan anesteziklere. </li><li> Ağızdan sonda yoluyla mısır yağı içinde çözülmüş CCl4 (2 ug / g, St Louis, MO) uygulayın. CCl4 uygulama prosedür IBC / EHS ilkelerine uygun olarak belirlenmiş bir bölge içinde yapılmalıdır. </li><li> 4 saat ve 24 saat sonra resmi. </li></ol></li></ol> 2. Bir Bioluminescent Görüntü Kazanılması <ol><li> Lüminesans moduna ayarlı IVIS 200 (Xenogen Corporation, Alameda, CA) Görüntüleme Sistemi, Initialize ve şarj bağlı cihaz (CCD) kilitlemek için sıcaklık için bekleyin. </li><li> Maruziyet süresi, binning (orta) ve F / Stop (8) ayarlarını seçin. </li><li> Normal vücut ısısını korumak için ateşli bir sahne görüntüleme odasında yatar pozisyonda yer fareler. Fareler IVIS görüntüleme odasında ise burun konisi ile 1.2 &#39;de tanımlanan anesteziklere. </li><li> L-012 yönetme<html> (20 mg / g) görüntüleme için seçilen her bir zaman noktasında retroorbital enjeksiyon yoluyla damardan steril PBS (10 mikrogram / ul) içinde eritildi. </li><li> L-012 enjeksiyonundan sonra 2 dk başlayarak görüntüler yakalayın. Biz genellikle 40 sn pozlama kullanın. </li></ol> 3. Region of Interest kullanarak Veri Analizi <ol><li> Yaşayan Image yazılımı V.4.2 (Xenogen Corporation, Alameda, CA) kullanılarak veriler analiz. </li><li> Bir görüntüyü açın ve İlgi araçları Bölgesi ölçümü seçin. </li><li> Göğüs ve / veya fare fotoğrafik görüntünün üzerinde pseudo-renkli dijital görüntü (foton algılama temsilen) overlaying sonra karın üzerine Faiz şekil ve boyut Bölge seçin. </li><li> İlgi Bölgesi&#39;nden gelen sinyal yoğunluğu (foton akısı) sayısal. </li></ol> 4. Istatistik <ol><li> Bireysel zaman noktalarında Bonferroni post-test ile iki yönlü ANOVA gerçekleştirmek için istatistiksel analiz yazılım paketi kullanın. </li></ol>tle &quot;&gt; 5. Temsilcisi Sonuçlar Zymosan bir pro-inflamatuar maya hücre duvarı ürünleri ve NADPH oksidaz 3 güçlü bir aktivatörü olan. Biz daha önce bu intratrakeal zymosan daha sağlam nötrofilik akciğer inflamasyonu ve S47 phox-/ pro-enflamatuar sitokin üretimini indükler gösterdi - farelerde 1 yabani-tip ile karşılaştırıldığında. Zymosan karşılaştırmadan sonra fareler - Burada amacımız yabani-tip ve S47 phox-/ akciğerleri ROS üretimi karşılaştırmaktır. 4 saat ve 24 saatte fareler - 4 saatte zymosan intratrakeal uygulamadan sonra, bu temel hem de S47 phox-/ göre yabani-tip olarak foton emisyon bir artış ile karşılaştırıldığında, yabani-tip farelerde göğüs boyunca foton emisyon önemli bir artış gözlenmiştir. Buna karşılık, S47 phox-/ in biyolüminesans sinyalleri - farelerde zymosan tedavisi (Şekil.1 AB) sonra 4 saatte bazal seviyelerde kalmıştır. Böylece, NADPH oksidaz b görünüyorE zymosan verilmesini takiben akciğerlerde ROS üretiminin ana kaynağı. Sonra, CLP-indüklenen sepsis modelinde ROS üretimi değerlendirilmiştir. Sepsis akut akciğer hasarı (ALI) ve akut solunum sıkıntısı sendromu (ARDS) 4, 5 gibi uç organ hasarı ile ilişkili hayatı tehdit eden bir sendromdur. ROS aracılı yaralanma sepsis kaynaklı çoklu organ disfonksiyonu sürüş önemli bir faktör olarak kabul edilmiştir. Biz ROS belirgin göğüs (4 saat) üzerinde ve başlangıca göre yabani-tip farelerden CLP takiben karın (24 saat) fazla arttığı bulunmuştur. Ancak, S47 phox-/ ROS seviyeleri - fareler zaman noktalarında (Şekil.2 AC) hem de bazal düzeyleri benzer bulunmuştur. Bu sonuçlar, CLP-indüklenmiş fare sepsis modelinde, ROS kuşak oksidaz bağımlı NADPH olduğunu göstermektedir. Son olarak, biz bir CCl 4-bağlı karaciğer hasarında modunda ROS üretimini tespit etmek bioluminescent görüntüleme kullanılırl. CCl 4 hepatosellüler nekroz neden olur ve akut karaciğer hasarı ve karaciğer fibrozisi 6 hem modellemek için kullanılır olmuştur. CCl 4 uygulamadan sonra 4 saatte fareler - iltihabı ve hasarı daha önce anlatılan modellerin aksine, biz karın ROS üretimi (yaklaşık% 35) S47 phox-/ başlangıca göre artış ılımlı olduğunu ortaya koymuştur. Fareler (Şekil 3 AB) - Ancak, 4-indüklenen ROS üretimi CCl büyüklüğünü S47 phox-/ daha yabani-tip farelerden anlamlı olarak daha fazla idi. Bu veriler hem S47 phox içeren NADPH oksidaz-bağımlı ve bağımsız ROS üretimi akut CCl 4-bağlı karaciğer hasarında meydana öneririz. <img alt="Şekil 1" src="/files/ftp_upload/3925/3925fig1.jpg" /> Zymosan tedaviden sonra akciğer Şekil 1. ROS üretimini. A) Temsilcisi bioluminescenROS üretimi ce görüntüleme. Fareler ve karın ışıldama zymosan muamele ile değişmez - göğüs ek olarak, lüminesans yabani-tip ve S47 phox-/ her ikisi de karın üzerine tespit edilebilir olduğuna dikkat edin. Yabani-tip ve S47 phox-/ ve göğüs B) foton sayım - zymosan bir (BT) intratrakeal tek enjeksiyondan sonra fareler. Biyoparlaklık görüntüleme taban, 4 saat ve 24 saat sonra yapıldı. Göğüs üzerinde ilgi bölgeden Işık emisyonu belirtilen sahte renk skalası kullanılarak tespit edilmiştir. Sonuçlar ortalama olarak sunulmaktadır ± SE, n = 6-9, grup başına * = p &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">büyük bir rakam için tıklayınız</a> . <img alt="Şekil 2," src="/files/ftp_upload/3925/3925fig2.jpg" /> Şekil 2. ROS tespiti sonrasında CLP. ROS üretimi, B A) Temsilcisi biyolüminesans görüntüleme) Foton göğüs sayıları ve yabani-tip ve S47 phox-/ ve karın C) foton sayımı - başlangıçta fare, 4 saat ve CLP sonra 24 saat. Sonuçlar ortalama olarak sunulmaktadır ± SE, n = grup başına 4-5, * = p &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">büyük bir rakam için tıklayınız</a> . <img alt="Şekil 3" src="/files/ftp_upload/3925/3925fig3.jpg" /> CCl sonrası karın 4-bağlı karaciğer hasarında Şekil 3. ROS üretimi. Oral CCl 4 tedaviden sonra başlangıçta fare, 4 saat ve 24 saat - ROS üretimi ve yabani-tip ve S47 phox-/ ve karından B) foton sayımı A) Temsilcisi biyolüminesans görüntüleme. . N = 4-5 grup başına, * = p &lt;0.05; Sonuçlar ortalama ± SE olarak sunulmuştur <a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">için tıklayınız büyük bir rakam</a> </a&gt;.

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Çıkar çatışması ilan etti.

Canlı hayvanlar reaktif oksijen türlerinin &quot;Gerçek zamanlı&quot; ölçümü (ROS) floresan ve kemilüminesans problar kullanılarak elde edilebilir. Floresan problar zayıf sinyal-gürültü oranı 12 olan muzdarip iken, açıklanan görüntüleme tekniği luminol tabanlı substrat L-012 ile ROS bir kimyasal reaksiyon sonrasında ışık emisyon tespiti için daha duyarlıdır. Tüm bioluminescent görüntüleme teknikleri gibi, bu metodoloji organ ve dokular tarafından dalgaboyu bağımlı ışık ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> L-012 </td><td> Wako Chemicals USA, Inc </td><td> 120-04891 </td><td></td></tr><tr><td> Zymosan </td><td> Sigma, St Louis, MO </td><td> Z4250 </td><td></td></tr><tr><td> karbon tetraklorid </td><td> Sigma, St Louis, MO </td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH oksidaz antibakteriyel ve antifungal konak savunma aracılık kritik bir enzimdir. Antimikrobiyal konak savunma rolüne ek olarak, NADPH oksidaz inflamatuvar yanıt 1 modüle kritik sinyalizasyon fonksiyonları vardır. Böylece, NADPH oksidaz kökenli ROS nesil &quot;gerçek zamanlı&quot; kinetik ölçmek için bir yöntem geliştirilmesi savunma, inflamasyon ve yaralanma barındırması ilgili mekanizmalar anlamak için değerli bir araştırma aracı olması bekleniyor. Kronik granülomatöz hastalık (CGD) ciddi enfeksiyonlar ve aşırı inflamasyon ile karakterize NADPH oksidaz kalıtsal bir hastalıktır. Fagosit NADPH oksidaz aktivasyonu, kendi sitosolik alt birimden (S47 phox, phox P67, P40 ve phox) ve bir membran-sınırlayıcı flavocytochrome (bir gp91 phox ve p22 heterodimeri phox oluşur) Rac translokasyon gerektirir. KayıpCGD bu NADPH oksidaz bileşenlerin sonucu herhangi birinde işlev mutasyonlar. ÇGD olan hastalarda benzer, gp91 phox-eksik farelerde ve S47 phox-eksik farelerde arızalı fagosit NADPH oksidaz aktivitesi ve bozulmuş konak savunması 13, 14 var. Yukarıda tarif edilen NADPH oksidaz bileşenler içeren fagositler, ek olarak, diğer hücre tipleri arasında, çeşitli NADPH oksidaz izoformları değişik ifade eder. Burada yaşayan farelerde ROS üretimini ölçmek ve inflamasyon ve yaralanma modellerinde ROS nesil NADPH oksidaz katkısını belirginleştiren bir yöntem açıklanmaktadır. Bu yöntem, bir şarj bağlı cihaz (CCD) tarafından kaydedilir ışıldama yayma L-012 (luminol bir analog) ile reaksiyona ROS dayanmaktadır. L-012 prob orijinal tanımı olarak, L-012-bağlı kemiluminesans tamamen bu reaksiyon tespit ana ROS süper olduğunu gösteren, süperoksit dismutaz kaldırılmış;oksit anyon 15. Daha sonraki çalışmalar, L-012 reaktif nitrojen türleri 16, 17 gibi diğer serbest radikaller, tespit edebildiğini göstermiştir. Kielland ve ark. 17 forbol miristat asetat, NADPH oksidaz güçlü bir aktivatörü, bu topikal uygulama ışıldayan sonda L-012 fare kullanılarak tespit edilebilir oksidaz bağımlı ROS NADPH üretim yol göstermiştir. Bu modelde, L-012-bağımlı lüminesans S47 phox-eksik farelerde kaldırılmış olduğunu gösterdi. Biz yabani-tip farelerden ve NADPH oksidaz eksikliği S47 phox-/ ROS üretimi karşılaştınl - aşağıdaki üç model fareler 2: zymosan, NADPH oksidaz aktive edebilirsiniz duvara türetilmiş ürün pro-inflamatuar fungal hücre 1) zerk; 2) çekal ligasyon ve delme (CLP), sekonder akut akciğer inflamasyonu ve hasarı ile intra-abdominal sepsis modeli ve 3) sözlü karbon tetraklorür(CCl4), ROS-bağımlı karaciğer hasarının bir model. Bu modeller özellikle sırasıyla, non-enfeksiyöz inflamasyon, polimikrobiyal sepsis ve toksin kaynaklı organ hasarının bağlamında NADPH oksidaz bağımlı ROS üretimi değerlendirmek için seçilmiştir. S47 için yabani-tip farelerde biyolüminesans karşılaştırma phox-/ - farelerde bize bu modellerde bioluminescent sinyal S47 phox içeren NADPH oksidaz tarafından üretilen ROS özgül katkıda tasvir sağlar. S47 ile karşılaştırıldığında yabani-tip farelerden artmış ROS seviyeleri gösterdi Biyoparlaklık görüntüleme sonuçları phox-/ - farelerde NADPH oksidaz enflamatuvar uyaranlara yanıt olarak ROS üretiminin ana kaynağı olduğunu belirtti. Bu yöntem, in vivo enflamasyon sırasında ROS nesil &quot;gerçek zamanlı&quot; izlenmesi için minimal invaziv bir yaklaşım sağlar.

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oksidaz fagositler reaktif oksijen türlerinin ana kaynağı (ROS) &#39;dir. Çünkü ROS geçici doğası nedeniyle, canlı hayvanlarda ROS seviyelerini ölçmek ve izlemek zordur. Yaşayan farelerde ROS seri ölçümü için bir minimal invaziv bir yöntem tarif edilmiştir.

Farklı Hayvan Modelleri NADPH Oksidaz Aktivitesinin Biyoparlaklık Görüntüleme

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

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Immunology and Infection

16.2K Görüntüleme.  Vanderbilt University School of Medicine. NADPH oksidaz fagositler reaktif oksijen türlerinin ana kaynağı (ROS) 'dir. Çünkü ROS geçici doğası nedeniyle, canlı hayvanlarda ROS seviyelerini ölçmek ve izlemek zordur. Yaşayan farelerde ROS seri ölçümü için bir minimal invaziv bir yöntem tarif edilmiştir.

Video: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Vivo Çift Yüzey Bioluminescent Imaging&#39;de

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Tıp

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

İzole Biventriküler ve NADH Floresans Görüntüleme Tavşan Kalpler Çalışma

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

İmmünoloji ve Enfeksiyon

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

BRET Ötesinde Bir Adım: Sınırsız Uyarma tarafından Flüoresan Lüminesans dan (YAKIT)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Biyomühendislik

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo Görüntüleme Yöntemi Akut ve Kronik İltihap ayırt

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Hem oksijenaz-1 ekspresyonunun Gua Sha Usul Biyoparlaklık Görüntüleme

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

Biyoloji

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

Biyolüminesan Optogenetik 2.0: Fotosensör Proteinleri In Vitro ve In Vivo'yu Aktive Etmek için Biyolüminesanstan Yararlanma

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Biyokimya

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Murin Nörogenezi Sırasında Çentik Sinyal Dinamiğinin Gerçek Zamanlı Biyolüminesans Görüntülemesi

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Gelişim Biyolojisi

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Bioluminescent Kalsiyum Göstergesi GFP-aequorin dayanarak Vivo Fonksiyonel Beyin Görüntüleme Yaklaşım

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Nörobilim

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

Bir taranabilir<em&gt; İn Vivo</em&gt; Transjenik biyolojik olarak ışık veren kullanarak Mitokondriyal Modülatörleri için tahlil<em&gt; Caenorhabditis elegans</em

Farklı Hayvan Modelleri NADPH Oksidaz Aktivitesinin Biyoparlaklık Görüntüleme

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