Yazarlar, İsveç araştırma konseyi, İsveç temel stratejik araştırma, İsveçli kanser toplum, İsveçli çocuk kanser toplumu, Åke WIBERGS vakıf ve Jeanssons vakıf tarafından desteklenmektedir.

1. Probu hücrelere Tanıtımı <ol><li> FRET-temelli bir prob ve bir plazmid görüşmekte direnci ile birlikte transfect hücreleri. Ilgi hücre tipi verimli bir transfeksiyon yöntemi seçin. U2OS hücreler için, standart kalsiyum fosfat transfeksiyon yöntemler yeterli sonuç verir 15. </li><li> En az yedi gün süreyle uygun antibiyotik hücreleri seçin. Bu zenginleştirir prob ve sınırları ciddi hücre döngüsünü etkileyen toksik ifade seviyeleri ya da ekspresyon düzeyleri ile hücrelerin miktarını ifade hücrelerin miktarı. </li><li> (Isteğe bağlı) istikrarlı klonlar için seçin. </li><li> Sabit veya canlı hücreler kullanarak, hem OBP ve YFP yaklaşık aynı oranda bütün hücrelerde mevcut olduğunu doğrulayın. </li><li> Bazı hücreler sadece CFP veya YFP içeren durumunda, muhtemelen rekombinasyon meydana geldi. Rekombinasyon bakteriyel büyüme sırasında veya prob transfeksiyon sonra da ortaya çıkabilir ve ikinci durumda plazmid kalite bağlı olarak değişebilir. Plazmid p tekrarlayınonarım ve sorun devam ederse transfeksiyon önce plazmid linearize. </li></ol> 2. İzleme oranlı metrik FRET <ol><li> Tohum hücrelerinin bir oda montaj için cam alt yemekler, veya kapak fişleri. Tabaklar / kapak fişleri hiçbir 1.5 (170 mikron kalınlığında) olduğunu emin olun. </li><li> 37 ° C&#39;ye kadar sıcaklık kontrolü ile motorlu Epifloresans mikroskop fenol kırmızısı olmadan Mount besi hücreleri PH düzenleme, medya ya da CO 2 veya CO 2-bağımsız medya gibi Liebowitz-15 olarak kullanarak emin olun. </li><li> Iletilen ışık kullanarak hücrelerin odaklanın. Floresan ışığı kullanarak hücreleri üzerinde bakmayın. </li><li> YFP uyarma (YFPex) ve YFP emisyon (YFPem) filtreleri ve uygun bir dikroik ayna kullanarak 12 veya 16 bit görüntü satın alarak başlayın. Hala gerekli Subselüler çözünürlük izin maksimal binning kullanın. Maruz kalma süresi ya da şiddeti azaltmak için yüksek bir binning fototoksisite önlemek için çok önemlidir. </li><li> Banaaşure veya hücrelerinden yoksun bir alanda kaba bir ortalama, minimum ve maksimum piksel değerleri ölçmek veya tahmin edilerek arka plan yoğunluğu ve yaklaşık gürültü tahmin. Değişim maruz kalma süresi ve nötral yoğunluk filtreleri hücre ortalama sinyal yaklaşık arka plan yoğunluğu artı 5-10 kez max ve min arka plan şiddetleri arasında fark olduğunu. </li><li> Doymuş olan yakın olmadığını görüntüler doğrulayın. Görüntüde maksimum yoğunluğu hücrelerin mitoz girdiğinizde yoğunluk farkları için izin vermek için dinamik aralık (örneğin bir 12 bit görüntü max 2000) yarısından daha az olmalıdır. </li><li> Adım 2,4-2,6 CFP uyarma ve YFP emisyon filtreleri ve uygun bir dikroik ayna kullanarak tekrarlayın. </li><li> Transfekte hücreler ile 2.7 ila 2.4 için kullanılan bölgeler hariç, birden çok bölge seçin ve bu maksimum yoğunluğu sağlamak tüm hücrelerde dinamik aralık yarısından daha az. </li><li> Iki görüntü her 4 edinme time-lapse Denemeyi başlatYFPex kullanarak 5 dakika - YFPem ve CFPex - YFPem filtre setleri. </li></ol> 3. Koşulları doğrulanıyor ve optimize <ol><li> Elde edilen görüntüleri açın ve mitoz transfekte sonda ifade hücrelerin mitoz hücre döngüsü süresi izlemek. </li><li> Ölçülen hücre döngüsü süresi, kullanılan hücre tipi için yayınlanmış veriler karşılaştırın. Alternatif olarak, film, referans hücre döngüsü kez almak için sadece iletilen ışık bir görüntü (örneğin, DIC veya faz kontrast) her saat satın alarak hücreleri untransfected. </li><li> Mitoz bölünme ile mitoz hücre döngüsü zamanlamaları referans karşılık durumda, 4. adıma geçin. Alternatif olarak, yeniden kazanmak zor maruz kullanarak görüntü, daha fazla zaman noktaları veya birden fazla z düzeyleri ve 3. adımı tekrarlayın. </li><li> Hücre döngüsü zamanlamaları referans karşılık gelmez mitoz bölünme ile mitoz durumda, filmin sonunda sadece iletilen ışık bir görüntü (örneğin, DIC veya faz kontrast) her saat bir görüntü elde hücreleri transfektetransfekte hücreleri belirlemek için deney. </li><li> Transfekte hücreler hala uzun bir hücre döngüsü kez göstermesi durumunda, 1. adımı tekrarlayın fakat daha az prob transfect veya prob çok düşük miktarda içeren kararlı klonlar için seçin. </li><li> Transfekte hücreler floresan ışık yokluğunda normal hücre döngüsü kez göstermesi durumunda, görüntüleri arasında binning, nötral yoğunluk filtreleri, maruz kalma süresi ve zamanı değiştirerek adım 2 ancak düşük maruziyet tekrarlayın. </li></ol> 4. FRET analiz <ol><li> Mikroskopi için adanmış birçok yazılım tarafından analiz yapılabilir. Burada ücretsiz ImageJ (kullanarak analiz yapmak için nasıl tarif <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a> eklenti Oranı Plus (kullanan) <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus html</a> ). </li><li> ImageJ görüntüleri açın. Durumda da görüntüler çok renkli Stac formattak (örneğin Deltavision) ilk kez bir hyperstack onları dönüştürerek tek tek kanallarını da ayrı: hyperstack Görüntü hyperstack yığını ve daha sonra onları kullanarak tek kanal yığınlarını bölünmüş: boyutluluk Görüntü hyperstack azaltmak </li><li> Bu adım sadece oranları, 0 ve 1 aralığında olmadığını sağlayarak görüntülerin görsel netlik artırmaya yönelik olarak, isteğe bağlı Süreç Matematik-çarpın: YFPem yığını - YFPex 3 ile çarpın . </li><li> YFPex - YFPem yığını, hücrelerinden yoksun bir alana ilgi bir bölge (ROI) çizmek, ancak bu ölçülebilir bir hücreye yakındır. Aynı görüntünün farklı hücreler, arka plan ve sinyal yoğunluğu hem de tipik bir görüntünün merkezinde güçlü olarak farklı bölgelerde gerekebilir. </li><li> ROI yöneticisi ROI ekle: Analiz-araçlar-ROI yöneticisi. </li><li> Analiz-Tedbir: - YFPem ve CFPex YFPem yığını YFPex hem de yatırım getirisi ortalama yoğunluğu ölçün </em&gt;. </li><li> Birden fazla timepoints tekrarlayın ve yakın ilgi hücre arka plan şiddetleri film boyunca benzer doğrulamak. </li><li> Minimum yoğunluğu ölçümleri analiz-Set-Min ve Max gri değeri: ölçümleri ayarlayın. </li><li> Ilgi bir hücrenin çoğunu kapsayan bir bölge çizin ve YFPex hem de en az yoğunluğu ölçmek - YFPem ve CFPex - YFPem yığını. 4.6 ölçülen minimum yoğunluğu ve arka plan yoğunluğu arasındaki fark atın ve ikiye bölün. Bu kırpma değeri için bir başlangıç ​​tahmin sağlar. </li><li> Açık Oranı Plus. CFPex ve YFPem - YFPem yığını YFPex seçin. Stack 1 olarak YFPem oranı seçin CFPex FRET - - yığını 1 olarak YFPem, ters fosforilasyon üzerine düşürür.Düzeltme FRET probları seçin YFPex görselleştirme oranı FRET için. </li><li> Ölçülen arka plan yoğunlukları ve kırpma değerleri yerleştirin. </li><li> Ölçekleme oranı yığını uygun bir göz u ayarlayın ve uygulayınp oranı değişiklikleri görmenizi tablosu (LUT). </li></ol> 5. Temsilcisi Sonuçlar Plk1 faaliyet mitoz sırasında G2 faz ve zirveleri çekirdeğinde ilk görünür. Şekil 3, 2 numaralı bölümde açıklandığı gibi, minimal fototoksisite ayarları kullanarak bir deney gösterir. Lütfen bu temsili bir ilk sonucu olduğuna dikkat ve maruz kalma koşulları veya görüntüleri arasında zaman sinyal gürültü oranı ya da zamansal çözünürlüğü artırmak için modifiye edilebilir. Prob ifade eden hücrelerin büyük bir çoğunluğu, 20 ila 25 saat arasında bir hücre döngü süresi ile görüntüleme koşulları ve prob ekspresyon düzeyleri hücre döngüsü zamanlamaları etkilemez belirten çoğaldığını Şekil 3D gösterir. Önemli gürültü olmasına rağmen, Plk1 aktivite trendi artan G2 ve mitoz 14 pik açıkça görülebilir (Şekil 3A). Burada, ham verilerin işlenmesi anlamına gelir ortalama ters ra kymograph (Şekil 3B) veya miktar olarak sunulan filtreleme<html> tio (Şekil 3C) netlik artırabilirsiniz. <img alt="Şekil 1" src="/files/ftp_upload/3410/3410fig1.jpg" /> Şekil 1 FRET tabanlı prob Prensibi kinaz ve fosfataz faaliyetlerini izlemek için. İki fluorophores, genellikle CFP (mavi) ve YFP (yeşil), fosfor bağlayıcı bir etki alanı (turuncu) ve phosphorylatable dizisi (sarı) bağlanır. Böylece prob bir konformasyonel değişikliği indükleyen fosfor bağlayıcı etki alanı bağlayıcı Fosforilasyon (kırmızı) aracılık,. OBP ve YFP arasında FRET verimliliği etkileyen iki fluorophores arasındaki mesafe veya yönde bir fark konformasyonel değiştirme sonuçları. FRET, heyecan verici OBP ve izleme YFP emisyonu (noktalı çizgiler) tarafından görüntülenmiştir olabilir. <img alt="Şekil 2" src="/files/ftp_upload/3410/3410fig2.jpg" /> Şekil 2 deneysel işlemin şematik anahat. p_upload/3410/3410fig3.jpg &quot;/&gt; Şekil 3 Plk1 faaliyet mitoz sırasında G2 faz ve zirveleri çekirdeğinde ilk görünür. 20x NA 0.7 hava objektif ve cıva lambası ile donatılmış bir Deltavision Spectris Görüntüleme sistemi kullanılarak 60 saat süreyle bir FRET tabanlı prob izleme Plk1 aktivitesi 9,14 ifade U2OS hücreleri çekildi. Görüntüleme koşulları 4x binning ve nötral yoğunluk filtreleri kullanarak, 2. bölümünde belirtildiği gibi, minimal fototoksisite neden seçildi gelen ışığın% 99 blok. 4 bölünmelerin bir hücre ters FRET-oranı, yanlış renk gösterimi,. B, ortalama bir filtre uyguladıktan sonra A gösterilen hücre kymograph. 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Biz ifşa etmek başka bir şey var.

Hücre döngüsü boyunca FRET izlenmesi, kısa vadeli dış uyaranlara tepkilerin değerlendirilmesi daha az önemli olan hususlar gerektirir. İlk olarak, hücre döngüsünün ilerlemesi kolayca bu fototoksisite minimumda tutulması gerektiren stres sinyalizasyon tarafından rahatsız edilir. İkincisi, tüm gazetecilere potansiyel kinazlar, fosfatazlar veya etkileşim alanları titre edilerek hücresel süreçleri etkileyebilir. Deneysel koşullar yeterli olup olmadığını değerlendirmek için belki de en basit y...

<table border="1"><tbody><tr><td> Reaktif </td><td> Katalog Numarası </td><td> Şirket </td></tr><tr><td> Leibovitz L-15, herhangi bir fenol kırmızısı </td><td> 21083-027 </td><td> Gibco, Yaşam Teknolojileri tarafından </td></tr><tr><td> DMEM + Glutamax-I </td><td> 31966 </td><td> Gibco, Yaşam Teknolojileri tarafından </td></tr><tr><td> Fetal sığır serumu (FBS) </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> % 0.05 tripsin EDTA </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td> Penisilin-Streptomisin </td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> Gibco, Yaşam Teknolojileri tarafından </td></tr><tr><td> Puromycin </td><td> P8833 </td><td> Sigma-Aldrich </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Förster rezonans enerji transferi (FRET) tabanlı gazetecilere 1 canlı hücrelerde bulunan endojen kinaz ve fosfataz faaliyetlerinin değerlendirilmesine izin verir. Bu problar genellikle phosphorylatable dizisi ve fosfor bağlayıcı etki alanı tarafından müdahale, OBP ve YFP türevleri oluşur. Fosforilasyon üzerine bir değişikliğe yol OBP ve YFP arasındaki mesafe veya yön değişikliği prob değişiklikler konformasyon, verimliliği FRET (Şekil 1). Birkaç probları PKA 2, PKB 3, KZÇ 4, 5 PKD, ERK 6, JNK 7, Cdk 18, Aurora B 9 gazetecilere ve Plk1 9 dahil olmak üzere birden fazla kinazlar ve fosfatazların faaliyet dengesi, izleme, son on yıl içinde yayınlanmış olan . Modüler tasarımı göz önüne alındığında, ek problar, yakın bir gelecekte 10 ortaya çıkması muhtemeldir . Hücre döngüsü boyunca ilerlemesi, stres signali etkilenirng 11 yolakları. Özellikle, hücre döngüsü, hücrelerin strese 12 kurtarma ile karşılaştırıldığında büyüme soğukkanlı sırasında farklı düzenlenir. Hücrelerinin hücre döngüsü boyunca Time-lapse görüntüleme, bu nedenle özel dikkat gerektirir. Oranlı metrik görüntüleme istihdam, özellikle yüksek sinyal gürültü oranı ile iki görüntü sonuçlarını doğru yorumlamak için gereklidir, çünkü bu bir sorun haline gelir. Oranlı metrik kinaz ve fosfataz aktiviteleri hücre döngüsü bağımlı değişiklikler görüntüleme ağırlıklı hücre döngüsü 8,9,13,14 alt bölümlerine sınırlı olmuştur FRET. Burada, insan hücre döngüsü boyunca oranlı metrik görüntüleme kullanarak FRET tabanlı probları izlemek için bir yöntem tartışın. Bu yöntem, pek çok araştırmacı, yaşam bilimleri ve mikroskopi veya görüntü işleme uzman bilgisi gerektirmez ekipman dayanmaktadır.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRET tabanlı gazetecilere giderek canlı hücre kinaz ve fosfataz aktiviteleri izlemek için kullanılır. Burada hedef fosforilasyon hücre döngüsü bağımlı değişiklikleri değerlendirmek için FRET tabanlı gazetecilere nasıl kullanılacağı hakkında bir metodu tanımlar.

Hücre Döngüsü FRET oranlı metrik sayesinde Kinaz ve Fosfataz Aktiviteleri İzleme

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

monitoring-kinase-phosphatase-activities-through-cell-cycle

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JoVE Journal

Biology

14.2K Görüntüleme.  Karolinska Institutet. FRET tabanlı gazetecilere giderek canlı hücre kinaz ve fosfataz aktiviteleri izlemek için kullanılır. Burada hedef fosforilasyon hücre döngüsü bağımlı değişiklikleri değerlendirmek için FRET tabanlı gazetecilere nasıl kullanılacağı hakkında bir metodu tanımlar.

Video: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Safen Ven ve Kardiyak Delinme Ratlarda Kan Alma

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Biyoloji

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Kabuklu uzun vadeli Lethal Toksisite Testi Artemia Franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

Unimoleküler Biyosensörler kullanma Canlı Hücreler Olaylar Sinyal gerçek zamanlı izlenmesi için Mikroskopi FRET

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Tek Molekül FRET DNA döngü oluşturma incelenmesi

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Senkronizasyon<em&gt; Caulobacter crescentus</em&gt; Bakteriyel Hücre Döngüsü Soruşturma

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Deneysel Yaklaşımlar Mitokondriyal Yerelleşme ve Nükleer Hücre Döngüsü Kinaz, Cdk1 Fonksiyonu Eğitim için

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

G Protein-seçici GPCR konformasyoları Canlı Hücre Süspansiyon Fluorometer Deneyinde FRET sensörleri kullanılarak ölçülebilir

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Mitotik hücre eşitleme ve yüksek çözünürlüklü Confocal mikroskobu mitoz sırasında çok fonksiyonlu hücre döngüsü proteinlerin rolü çalışmaya birleştirme

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...