Biz kritik bir yorum ve sorun giderme günlük bazda photobleaching deneyler için, özellikle Meshorer laboratuar üyeleri, Shai Melcer, Adi Alajem, Edupuganti Raghu Ram, badi Sri Sailaja, Anna Mattout ve Alva Biran teşekkür ederim. EM, Joseph H. ve Belle R. Braun Yaşam Bilimleri Kıdemli Öğretim Üyesi ve İsrail Bilim Vakfı (ISF 943/09), İsrail Sağlık Bakanlığı (6007) Avrupa Birliği (IRG-206.872 ve 238.176) tarafından desteklenen İsrail Kanser Araştırma Vakfı, İbrani Üniversitesi ve İsrail Psikobiyoloji Enstitüsü İç uygulamalı Tıp Hibeler.

1. ES Hücreler Kaplama T = 0 sa MEF kaplama <ol><li> Tabakanın canlı görüntüleme μ-Slaytlar 8-kuyu (ibidi; Münih, Almanya), jelatin veya odacıklı kapak camları (Lab-Tek, Rochester, NY) ile veya cam alt kültür yemekleri (Mattek; Ashland, MA). 5-30 dakika bekletin ve ücretsiz jelatin uzak aspirat. </li><li> Tohum 22.000 MEFS / DMEM 250 ul toplam hacmi [% 10 fetal sığır serumu (FBS) ile desteklenmiş]. Hücreler doku kültürü inkübatör (37 ° C,% 5 CO2) büyümesine izin verin. </li></ol> T = 6 saat ES hücre kaplama <ol start="3"><li> Aspire DMEM. </li><li> Tohum / ES hücre medya 250 ul toplam hacmi 15.000 R1 hücreleri ile kaplı her MEF [% 10 ESC dereceli fetal sığır serumu (FBS), 1 mM sodyum piruvat, 0.1 mM aminoasitlerin, 0.1 mM β-ile takviye merkaptoetanol, ve 1000 U / ml lösemi inhibitör faktör (LIF)], ertesi gün% 30 -% 50 izdiham elde etmek için. </li></ol> 2. ES Hücreler Transfecting T = 24 saat Geçici transfeksiyon <ol><li> 250 ul / kuyu taze ES hücre medya ES hücre ortamı değiştirin. </li><li> 1.5 ml steril test tüpü, 100 ul serum ücretsiz medya [Opti-MEM (Gibco)], serum serbest ortam içine doğrudan 10 ul transit LT1 transfeksiyon reaktifi (Mirus) ekleyin. Nazik pipetleme ile karıştırın ve 5-20 dakika oda sıcaklığında inkübe edin. </li><li> 1.5 mg GFP füzyon plazmid DNA (H1e, H1o veya HP1) seyreltilmiş transit-LT1 reaktif ekleyin. Nazik pipetleme ile karıştırın ve 15-30 dakika oda sıcaklığında inkübe edin. </li><li> 13.5 ul / kuyu transfeksiyon karışımı ekleyin. Swirl μ-Slaytlar 8-hatta dağılma sağlamak için iyi. 24 saat 250 ul taze ES hücre medya ile eski ES hücre medya değiştirdikten sonra. </li></ol> 3. Sıkı bağlamak ve FLIP Sahne T = 48-72 saat <ol><li> Deneyde herhangi bir konfokal lazer tarama mikroskobu (CLSM) yapılabilir, ancak normal bir sıkı bağlamak / FLIP deneyde bu yana, birçok üst üste görüntüleri elde toplama hızı sağlayan dönen bir disk konfokal mikroskop kullanmak için önerilen ve sağlar dair hiçbir beyazlatma istenmeyen numune ilk kasıtlı beyazlatma olay meydana gelecektir. Burada, Yokogawa CSU-X dönen disk kafası disk konfokal sistemi (www.Andor.com) Devrim iplik kullanmanızı öneririz. Bu sistem bir nokta tarama sistemi ile özel bir FRAPPA modülü kullanarak photobleach çift kapasiteye sahip ve hızla dönen disk kullanarak görüntü toplamak için ışık geri dönmek. Deneyler photobleaching için kullanılan en yaygın 3 floresan proteinleri GFP, YFP ve Kiraz. GFP veya YFP kullanılırsa, bir ~ 488 nm lazer gereklidir. Kiraz, ~ 560 nm lazer kullanın. Tüm durumlarda, katı hal lazerleri kullanmanızı öneririz. Otomatik bir aşamada olması yararlı ancak gerekli değildir. Canlı hücreler görüntülü olduğundan, (LIS gelen birini kullanıyorsanız, İsviçre) bir kazanı kullanmak için gerekli oksijeni kontrol, nem, CO 2 ve sıcaklık. Sıkı bağlamak gerekli minimal lazer gücü (genellikle% 10 alan, floresan seviyesi yeterli) ile görüntüleme yapılırken maksimum lazer yoğunluğu kullanılarak yapılır. Uygun dalga boyu floresan ışık hücreleri gözlemleyin ve 60X immersiyon yağı lens kullanarak GFP ifade eden bir hücreyi seçin. Doğru hücre içi dağıtım olun. Bazen, ekspresyon düzeyleri çok yüksek, protein lokalizasyonu nükleolus gibi diğer bölmelere &#39;dökülme&#39;. Bu tür hücreler seçili olmamalıdır. </li><li> Şimdi bir görüntüleme protokolü ayarlayın: ökromatin veya heterokromatin (yoğun GFP odakları olarak görülüyor) sonra, photobleaching önce photobleach 3-5 kare toplamak ve 250-1000 ms aralıklarla, photobleach sonra 90-120 kare toplamak: H1e-GFP, 1000 ms H1o ve HP1-GFP, 250 ms (zaman aralığı değişiklikleri son derece dinamik proteinler, kısa bir zaman aralığı gerektiren protein dinamikleri, göre). Biz normalde 80-100 lazer yoğunluğu% 20-40 μseconds (1-2 tekrarlamalar) bir lazer darbesi ile photobleaching kullanır, ancak bu sayılar, analiz edilen protein ve ekspresyon seviyeleri göre değiştirebilirsiniz. Photobleaching uygun olduğunda, GFP floresan bir &quot;kara delik&quot; gözlemlemek gerekir. Kara delik giderek floresan aşağıdaki kurtarma yeniden doldurulur. Dönen disk, saniyede 60 görüntü (iyi floresan ve tek bir hücre üzerinde yakınlaştırma) elde edebilirsiniz rağmen, biz, düşük görüntü kalitesi ve potansiyel artışı fototoksisite sayesinde yüksek hızlarda sistem kullanmanızı tavsiye etmiyoruz. </li><li> FLIP bir deneme için, farklı bir görüntüleme protokolü kurmak, ağartma önce 3-5 kare toplamak sonra görüntüleri toplarken aynı noktada beyazlatma tekrarlanmalıdır. H1e H1o-GFP, çamaşır suyu, GFP, çamaşır suyu, her 5 saniye, her 2 sn ve HP1-GFP çamaşır suyu her 1 sn için. Bleach bird tüm deney boyunca tekrar tekrar görüntüleri toplamak. </li><li> Her iki teknik için, 20-30 hücreleri üzerinde işlemi tekrarlayın. Istatistiki amaçlar için, tercihen farklı günlerde, deney 3 veya daha fazla kez tekrarlayın. Homojen bir nüfus ve doğru ayarları, standart sapma, genellikle düşük (&lt;% 5). Hem sıkı bağlamak ve FLIP için, ağartılmış bölgenin büyüklüğü ve şekli kurtarma dinamiklerini etkiler ve bir deney içinde sabit kalmalıdır. Ayrıca, iki hücre karşılaştırıldığında, aynı protokolleri kullanılan olmalıdır ve hücrelerin güç lazer ve diğer koşulları dalgalanma gösterebilir ve deney sonucunu etkileyebilecek aynı gün sırayla analiz edilmelidir. </li></ol> 4. Sıkı bağlamak ve FLIP Veri Analizi <ol><li> Tüm sıkı bağlamak kare toplanan, floresan yoğunluğu ölçümü ROI (yatırım getirisi b = ağartılmış alan), arka plan alanı (ROI bg) ve önce zamanın bir fonksiyonu olarak ve ağartma sonra olmayan ağartılmış nükleer alanı (ROI nb). Ağartılmış bölgenin ihmal olduğunda tüm çekirdek normalleşme amaçlar için seçilebilir. </li><li> / (ROI nb - ROI bg) - (ROI bg ROI b) / (pbROI b - pbROI bg) / (pbROI nb - pbROI bg), pb önceden ağartılmış ifade eder: her zaman noktası için, formüle göre veri normalleştirmek. -Bleach öncesi görüntüleri için yaklaşık 1 bir değer almalısınız. Çamaşır suyu sonra ilk görüntü Bleach Derinlik gösterecektir. Gerçek Bleach Derinlik değeri için 1 değeri çıkarma. Her hücre ve her bir deney ortalama 20-30 hücreleri için bu işlemi tekrarlayın. </li><li> Toplanan tüm FLIP kare, olmayan ağartılmış nükleer alanda floresan ve arka alanı (ROI nb = ağartılmış olmayan alan, YG bg = arka) ölçer. FLIP veri hesaplanması sıkı bağlamak eğrisine benzer, sadece analiz yatırım getirisi (ROI nb) hesaplanmasında kullanılan gerçek beyazlatılmış bölge, farklı olmalıdır: (ROI nb - ROI bg) / (pbROI nb - pbROI bg) . Normalleştirme amaçlı bir komşu hücre (ROI n = komşu hücre) kullanmak da mümkündür: (ROI nb - ROI bg) / (ROI n - ROI bg) / (pbROI nb - pbROI bg) / (pbROI n - pbROI bg .) Veri toplama, bilgisayar simülasyonu için deneysel veri sığdırmak mümkün. Bu iyi bir yakınlık, cep fraksiyonu, hareketsiz fraksiyonu ve yarı-maksimum, hesaplama sağlar. Biz burada sıkı bağlamak analizler matematiksel ve istatistiksel yönlerini tartışmak ve diğer mükemmel yayınlar 4-9 okuyucu bakın olmaz. Çamaşır suyu derinliği önceden çamaşır suyu (% 100), sinyal ve çamaşır suyu sonra ilk görüntü arasındaki mesafe (y ekseni) ifade eder; mobil fraksiyonu çamaşır suyu derinliği ve arasındaki mesafe (y ekseni) ifade eder Kurtarılan sinyal kinetik bir platoya ulaşır ve hareketsiz fraksiyonu kurtarılan sinyal ve ön çamaşır suyu (% 100) sinyal arasındaki mesafe (y-ekseni) (bkz. Şekil 1B ve 2B) ifade eder. Bu analizin ötesinde, veri sığdırmak için iyi bir matematiksel modeller vardır. Tek bir üs, denklem için  <img src="/files/ftp_upload/2696/eq0.jpg" alt="Denklem" width="80" /> t zaman nerede, A, 1-A mobil fraksiyonu hareketsiz fraksiyonu ve k off sabit ayrışma, veri sığdırmak için kullanılır ve kapalı bağlama oranı doğrudan tahmin edilebilir (k off) yanı sıra, dernek oranı hesaplamak için kullanılan parametre, elde. </li></ol> 5. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/2696/2696fig1.jpg" alt="Şekil 1" /> Şekil 1 A ve B gösterisi temsilcisi sıkı bağlamak eğrileri HP1 (solda), H1o (orta) ve R1 ES hücrelerinin H1e (sağda). Sadelik ve berraklık Şekil için 1A herhangi bir normalleşme ve hesaplama önce tek bir hücre ham verileri gösterir. Sarı eğrisi ağartılmış bölgeye karşılık, mor eğri olmayan ağartılmış nükleer alanı (ağartılmış bölgenin ihmal olduğunda tüm çekirdeği normalleşme amaçlar için seçilebilir) karşılık, kırmızı çizgi olan eşiğe karşılık gelen Bu durumda minimal. Dikey ok çamaşır suyu temsil eder. Normalize ve ortalama veriler Şekil 1B gösterilmiştir. H1 (mavi) HP1 (kırmızı) ile karşılaştırıldığında daha yavaş iyileşme unutmayın. Ayrıca H1e varyant (koyu mavi) H1o varyant (açık mavi) daha yavaştır. Mobil ve sabit kesirler ve Bleach Derinlik HP1 gösterilir. <img src="/files/ftp_upload/2696/2696fig2.jpg" alt="Şekil 2" /> 2. A ve B ökromatin karşılaştırarak göstermek temsilcisi sıkı bağlamak eğrileri (ŞekilR1 ES hücrelerinin HP1 heterokromatin (sağda) ile birlikte) bıraktı. Benzer şekilde Şekil 1, Şekil 2A, tek bir hücrenin ham verileri gösterir, sarı eğrisi ağartılmış bölgeye karşılık, mor eğri olmayan ağartılmış nükleer alana karşılık, kırmızı çizgi eşiğe karşılık gelir. Dikey ok çamaşır suyu temsil eder. Normalize ve ortalama veriler Şekil 2B gösterilmiştir. Heterokromatin (koyu kırmızı) ökromatin (açık kırmızı) ile karşılaştırıldığında daha yavaş iyileşme unutmayın. Mobil ve sabit kesirler ve Bleach Derinlik ökromatin gösterilir. <img src="/files/ftp_upload/2696/2696fig3.jpg" alt="Şekil 3" /> Şekil 3. H1o R1 ES hücreleri tipik bir FLIP deney Şekil 3A (ham, un normalize veri) ve B (normalize ve ortalama veri) gösterilir. Bu deneyde, mor eğri olmayan ağartılmış nükleer alana karşılık, yeşil hat komşu bir hücre çekirdeğine karşılık gelen ve kırmızı çizgi eşiğe karşılık gelir.

<ol>
	<li>Phair, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+nature+of+dynamic+protein-chromatin+interactions+in+vivo:+three-dimensional+genome+scanning+and+dynamic+interaction+networks+of+chromatin+proteins.">Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins.</a> Mol Cell Biol. 24, 6393-6402 (2004).</li><li>Meshorer, E., Girard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging chromatin in embyonic stem cells in StemBook. , (2008).</li><li>Meshorer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdynamic+plasticity+of+chromatin+proteins+in+pluripotent+embryonic+stem+cells.">Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.</a> Dev Cell. 10, 105-116 (2006).</li><li>Bancaud, A., Huet, S., Rabut, G., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photo conversion, and FLIP. , (2009).</li><li>Dundr, M., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+dynamics+of+nuclear+proteins+by+photobleaching.">Measuring dynamics of nuclear proteins by photobleaching.</a> Curr Protoc Cell Biol. Chapter 13, Unit 13-Unit 13 (2003).</li><li>Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+membrane+dynamics+and+reassembly+in+living+cells:+targeting+of+an+inner+nuclear+membrane+protein+in+interphase+and+mitosis.">Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis.</a> J Cell Biol. 138, 1193-1206 (1997).</li><li>Lenser, T., Weisshart, K., Ulbricht, T., Klement, K., Hemmerich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+fluctuation+microscopy+to+reveal+3D+architecture+and+function+in+the+cell+nucleus.">Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.</a> Methods Cell Biol. 98, 2-33 (2010).</li><li>Mueller, F., Mazza, D., Stasevich, T. J., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+and+kinetic+modeling+in+the+analysis+of+nuclear+protein+dynamics:+what+do+we+really+know.">FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know.</a> Curr Opin Cell Biol. 22, 403-411 (2010).</li><li>Phair, R. D., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modelling+approaches+to+in+vivo+imaging.">Kinetic modelling approaches to in vivo imaging.</a> Nat Rev Mol Cell Biol. 2, 898-907 (2001).</li><li>Poser, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+TransgeneOmics:+a+high-throughput+method+for+exploration+of+protein+function+in+mammals.">BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.</a> Nat Methods. 5, 409-415 (2008).</li><li>Sigal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+fluorescently+labeled+endogenous+protein+library+in+living+human+cells.">Generation of a fluorescently labeled endogenous protein library in living human cells.</a> Nat Protoc. 2, 1515-1527 (2007).</li><li>Cohen, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+proteomics+of+individual+cancer+cells+in+response+to+a+drug.">Dynamic proteomics of individual cancer cells in response to a drug.</a> Science. 322, 1511-1516 (2008).</li></ol>

Hücre popülasyonlarının veya sabit hücreleri, sıkı bağlamak deneyler saflaştırılmış kromatin içeren en uygun teknikler, aksine, canlı hücrelerde kromatin protein dinamikleri değişiklikler izleyin. Kromatin protein dinamiği kromatin plastisite için iyi bir gösterge olarak bulundu. Ancak, bu fırınlama, GFP ilgi geninin gerektirdiğinden, floresan etiketinin yanı sıra protein fonksiyonu ile karışabilir. Bu nedenle, sıkı bağlamak geçmeden önce, füzyon proteini titizlikle kendi doğal muhatab...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> DMEM </td><td> Sigma </td><td> D5671 </td></tr><tr><td> Jelâtin </td><td> Merck </td><td> 1,04078 </td></tr><tr><td> Opti-MEM </td><td> Gibco </td><td> 31985 </td></tr><tr><td> Transit LT1 </td><td> Mirus </td><td> MIR2300 </td></tr><tr><td> Μ-Slaytlar 8-iyi </td><td> ibidi </td><td> 80826 </td></tr></tbody></table>

photobleaching assays (frap &amp; flip) to measure chromatin protein dynamics in living embryonic stem cells

Photobleaching sonra Floresan Kurtarma (sıkı bağlamak) ve Photobleaching olarak Floresan Kaybı (FLIP) iyi mekansal ve zamansal çözünürlüğe sahip canlı hücrelerin protein dinamikleri çalışma sağlar. Burada sıkı bağlamak ve kromatin proteinleri, H1 ve HP1, fare embriyonik kök (ES) hücreleri de dahil olmak üzere FLIP deneyleri gerçekleştirmek için nasıl açıklar. Sıkı bağlamak deneyde, hücreler, yeşil floresan protein (GFP) veya bunların türevleri (YFP, CFP, Kiraz, vb.) Ile kaynaşmış bir ilgi protein, ya geçici ya da stabil bir şekilde, transfekte vardır. Transfekte floresanlama hücreleri, yoğun bir ilgi odaklı lazer ışını nispeten küçük bir bölge (ROI) beyazlatıcılar. Lazer dalga boyu, füzyon için kullanılan floresan protein göre seçilir. Geri dönüşümsüz yatırım getirisini lazer ışığı floresan sinyal moleküllerin beyazlatıcılar ve hemen beyazlatma aşağıdaki ağartılmış alan floresan sinyal kurtarma ağartılmamış molekülleri ile ağartılmış moleküllerin yedek aracılık zaman atlamalı görüntüleme ile izlenir. Üretilen floresan kurtarma eğrileri protein hareketlilik hakkında bilgi sağlar. Floresan moleküller hareketsiz iseniz, herhangi bir floresan iyileşme görülecektir. Tamamlayıcı bir yaklaşım, Photobleaching içinde Floresan Kaybı (FLIP), lazer ışını aynı noktaya tekrar tekrar beyazlatıcılar ve başka floresanlama hücre sinyal yoğunluğu ölçülür. Bu nedenle FLIP deneyler yerine floresan kurtarma ölçmek ve sinyal çürümesi, hücresel bölmeleri arasında mekik protein hareketlilik gibi, protein belirlemek için yararlıdır. Geçici bağlanma kromatin ile ilişkili proteinlerin ortak bir özelliği. Her kromatin protein kararlı durum herhangi bir anda büyük bir bölümü kromatin bağlı olmasına rağmen, bağlama, geçici ve en kromatin proteinleri saniye sırasına göre bir ikamet süresi, kromatin üzerinde yüksek bir ciroya sahip. Bu özellikler, yüksek plastisite genom ifade 1 üretmek için çok önemlidir . Photobleaching deneyler kromatin proteinleri (heterokromatinin protein 1 (HP1), bağlayıcı histon H1 ve çekirdek histon dahil) dinamik değişimi yüksek olduğu, bu nedenle özellikle ES hücrelerinin kromatin plastisite, kromatin yapısal proteinleri, GFP-füzyon sürümlerini kullanan belirlemek için özellikle yararlı 2,3 farklılaşmış hücrelerin daha.

Watch this Scientific Journal Video about Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells at JoVE.com

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Biz Photobleaching sonra Floresan Kurtarma (sıkı bağlamak) ve embriyonik kök hücreleri (ES) kromatin protein dinamiklerini izlemek için (FLIP) Photobleaching Floresan kaybı da dahil olmak üzere photobleaching yöntemleri açıklanmaktadır. Kromatin plastisite çalışma araçlarının biri olarak kabul edilir Kromatin protein dinamiği, pluripotent hücreler geliştirilmiş.

Embriyonik Kök Hücreler Yaşayan Kromatin Protein Dynamics Tedbir Tahliller (sıkı bağlamak ve FLIP) Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

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Biology

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

27.1K Görüntüleme.  The Hebrew University of Jerusalem. Biz Photobleaching sonra Floresan Kurtarma (sıkı bağlamak) ve embriyonik kök hücreleri (ES) kromatin protein dinamiklerini izlemek için (FLIP) Photobleaching Floresan kaybı da dahil olmak üzere photobleaching yöntemleri açıklanmaktadır. Kromatin plastisite çalışma araçlarının biri olarak kabul edilir Kromatin protein dinamiği, pluripotent hücreler geliştirilmiş.

Video: Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Bio Flip-Chip kullanarak Embriyonik Kök Hücreler, Desenlendirme

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

İnsan Embriyonik Kök Hücreleri Mitokondrial Kompleksi Aktivite araştırmayı

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

İnsan Embriyonik Kök Hücreler Donma ve Çözülme

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Embriyolar yılında Programlı Hücre Ölümü Algılama LysoTracker kullanımı ve Embriyonik Kök Hücreler Farklılaşan

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Kantitatif RT-PCR ile Bireysel İnsan Embriyonik Kök Hücreleri profilleme

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Içinde Mikrotubul Dynamics Tedbir plusTipTracker Yazılım Kullanma<em&gt; Xenopus laevis</em&gt; Büyüme Konileri

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...