Bu çalışma, (DEC) R35GM119617 vermek Thomas F. ve Kate Miller Jeffress Memoria (DEC) güven ve NIH tarafından desteklenmiştir. konfokal mikroskop görüntüleme VCU Nanomaterials Karakterizasyonu Çekirdek (KKK) Tesisinde gerçekleştirilmiştir.

1. Nesprin-2G Sensör DNA ve diğer plazmid DNA elde edilir <ol><li> ticari bir kaynaktan Nesprin-2G TS (gerilim sensörü), Nesprin-2G HL (başsız) kontrol, mTFP1, venus, ve TSmod elde edilir. Bütün DNA plazmidleri yaymak ve daha önce 12, 13 tarif edildiği gibi, örneğin DH5a-a gibi Standart E. coli suşları, kullanarak saflaştırılması. </li></ol> Nesprin-2G ve diğer Plazmid DNA ile 2. transfekte hücreler <ol><li> Sıcaklık (37 ° C) ve CO2 (% 5) düzenlenmesi ile standart bir hücre kültürü inkübatöründe bir 6-yuvalı hücre kültürü çanağı içinde% 70-90 konflüansa NIH 3T3 fibroblast hücrelerinin büyütün. hücre büyüme ortamı için,% 10 fetal dana serumu ile takviye edilmiş Dulbecco Modified Eagle Medium (DMEM) kullanın. </li><li> bir hücre kültür kaput, orta kaldırmak ve bir indirgenmiş serum hücre ortamında yaklaşık olarak 1 mL&#39;si ile de her yıkayın. Her bir oyuğa indirgenmiş serum hücre ortamında 800 mcLve kuluçka makinesi içinde 6 oyuklu odasına yerleştirin. </li><li> Lipid taşıyıcı çözeltisi 35 uL 1.5 ml tüp içine indirgenmiş serum hücre ortamında Pipet 700 uL &quot;lipomix&quot; oluşturulur. Pipetleme ile karıştırın. bir &quot;L&quot; ile tüp etiketleyin </li><li> Altı 1.5 ml tüpler toplayın ve her bir tüpe indirgenmiş serum hücre ortamında 6. pipetle 100 uL aracılığıyla onları 1 etiketleyin. <ol><li> mVenus 5. pipetle 1 ug tüp içine borular 3 ve 4 pipetle 1 OVMP ug içine tüpler 1 ve 2 Pipet 2 Nesprin-HL ug içine aşama 1, pipet 2 Nesprin 2G-TS ug plazmid DNA konsantrasyonu kullanılarak tüp 6. içine yeniden kullanım etmeyin pipet uçları DNA&#39;nın farklı türde pipetlerken zaman. </li></ol></li><li> Her bir işaretlenmiş borusu (1-6), &quot;L&quot; tüp lipomix Pipet 100 uL ve tekrar pipetleme karıştırın. Her tüp için temiz pipet kullanın. 10-20 dakika süreyle inkübe edin. </li><li> % 70-90 konfluent 6 oyuklu odası içinde bir kuyuya her etiketli tüp 200 mcLHücreler. karşılık gelen DNA ile her bir üst Etiket ilave edildi. 46 saat için bir kuluçka makinesi içinde 6 oyuklu odasına yerleştirin. </li><li> orta aspire ve durulama için indirgenmiş serum hücre ortamında 1-2 ml. , Indirgenmiş serum hücre ortamı aspire her bir göze tripsin 2 mL ekleyin ve inkübatör (5-15 dakika), 6-çukurlu bir tabak yerleştirin. </li><li> Hücreler inkübatör içinde ayırmak birlikte, kat 6 cam alt fosfat tamponlu tuzlu su (PBS) içinde çözülmüş 20 mg / mL&#39;lik bir konsantrasyonda fibronektinle bir tabaka ile bulaşıkları görüntüleme. kaplamak için yemeklere hücre kültürü kaputu (yaklaşık 20 dakika) yüzey izin verin. </li><li> Hücreler müstakil bir kez DMEM 2 mL eklenerek tripsin nötralize. </li><li> iyi bir etiketli 15-ml santrifüj tüpüne her içeriğini aktarın ve sallanan bir rotor santrifüj içinde 5 dakika için 90 x g&#39;da aşağı doğru döndürün. Süpernatant aspire ve pipet karıştırma ile DMEM 1.000 uL, her hücre topağı yeniden askıya. </li><li> fibronektin karışımı aspireCam kaplar ve pipet cam tabaklar üzerine her bir hücre süspansiyonu 1.000 uL. </li><li> Hücreler cam kasesi taban (~ 15 dakika) yerleşmek sonra hücre kuluçka makinesi içinde de yer ve her DMEM başka bir 1 ml +% 10 FBS +% 1 Pen-Strep ekleyin. Hücreler en az 18-24 saat sensör takmak ve ekspres izin verin. Not: Hücreler, ticari katyonik lipid transfeksiyon reaktifleri (Malzemelerin Tablo) kullanılarak transfekte edilir. Seçenek olarak ise, stabil hücre hatları, bir toksine karşı direnç kazandıran bir gen ile bir plasmid kullanılarak seçilebilir (Nesprin-TS ve -HL için pcDNA plazmidler genetisin direnci ifade eden hücreler (Malzemelerin Tablo) temin eden bir DNA depo web sitesinde bulunabilir ). Ek olarak, viral enfeksiyon yöntemleri (lentivirüs, retrovirüs, veya adenovirüs) transfekte etmek zor olan hücrelerde sensörü ifade etmek için kullanılabilir. </li><li> Nesprin-TS ek olarak, Nesprin HL sıfır için ek hücreleri transfektece kontrol adımlar 2.1-2.12 tarif edildiği gibi; TSmod aynı zamanda sıfır güç kontrol olarak kullanılabilir ve Nesprin-HL benzer FRET sergilemelidir. </li><li> mTFP1 ve venus ile transfekte hücreler spektral parmak izi (Adım 4) üretmek için. Not: mTFP1 ve venus, tipik olarak Nesprin-TS daha yüksek seviyelerde eksprese eder ve bu şekilde DNA alt miktarların benzer ekspresyon seviyelerini elde etmek için transfekte edilmesi gerekebilir. </li></ol> 3. Transfeksiyon Verimi doğrulama <ol><li> Yaklaşık 18-24 saat transfeksiyon tamamlandıktan sonra, görünümünde hücreler (genellikle% 5-30) toplam sayısı flüoresan hücrelerin sayısını karşılaştırarak transfeksiyon etkinliğini incelemek için bir ters flüoresan mikroskop kullanılır. <ol><li> 462 nm&#39;de (mTFP1) ya da 525 nm&#39;de (venus) ve 492 nm&#39;de (mTFP1) ya da 525 nm&#39;de (venüs) yakınında bulunan bir emisyon filtresi yakın bir uyarım frekansı ile donatılmış bir floresan mikroskobu kullanarak. NOT: Alternatif olarak, GFP filtre setleri mTFP1 bir arada yakalamak ve ettik edeceknus emisyon transfeksiyon verimliliği teyidini sağlayacak ve. </li></ol></li><li> Transfeksiyon sonrası 48 saat içinde resmi canlı hücreleri. <ol><li> Seçenek olarak ise, 48 saat 8 sonra, PBS içinde 5 dakika, deposu için, ve görünümü (kalsiyum ve magnezyumlu PBS içinde)% 4 paraformaldehid içinde hücrelerin tespit. NOT: 48 saat ötesinde, sinyal kalitesi ve gücü azalır. Hücrelerin Tespit 8 ifade edilen FRET dahil olmak üzere durumunu korur; orta montaj FRET 14 etkileyebilir olarak, ancak, hücrelerin sadece PBS içinde görüntülü olmalıdır. ekspresyonunda bir değişiklik olabilir gibi sabit hücreleri yalnızca, diğer sabit hücrelere kıyasla olabilir. Dikkat: Paraformaldehyde toksiktir. uygun kişisel koruyucu ekipman (KKE) giyin. </li></ol></li></ol> Spektral unmixing için mTFP1 ve Venüs floroforların 4. Yakalama Spektral parmak izleri <ol><li> Bir hücre kültürü kaputu, görüntüleme Mediu ile hücre orta yerine% 10 fetal sığır serumu ile takviye edilmiş m (HEPES-tamponlu). </li><li> sıcaklık-kontrollü bir (37 ° C), konfokal mikroskop aşamasında görüntüleme yemekler yerleştirin. </li><li> 1.4 olan bir sayısal açıklık ile 60X büyütmede yağ objektiv mTFP1-transfekte edilmiş hücreler ile cam görüntüleme tabağına yerleştirin. NOT: Yağ yakından cam zeminin kırılma indeksi benzemeye 60X objektif bir lazer tarama mikroskobu kullanılmaktadır. Yağ objektif lens üstüne yerleştirilir ve cam kapak ile doğrudan temas edilir. </li><li> mTFP1 bir 458 mm uyarım kaynağı ve 500 nm de bir emisyon bant-geçiş filtresi ile eksprese eden hücrelerin bulun. </li><li> Görüş alanında bir flüoresan hücre ile, (burada kullanılan yazılım &quot;Lambda modu&quot;) spektral algılama modu seçmek ve spektral görüntü (Şekil 3-1) yakalamak; 10 mil artışlarım (Şekil 3-3) kullanılarak 458 nm ötesinde tüm frekansları içerir. Parlak fluoresc seçinHücre (20 piksel uzaklık) üzerinde ent bölgesi (ROI). Not: mTFP1 eksprese ROI spektral şekil hücresi boyunca nispeten sabit kalmalıdır. şekil büyük ölçüde farklı ise, lazer yeniden ayarlayın ve sinyal-gürültü oranını geliştirmek için ayarları kazanır. </li><li> tıklayarak spektral veritabanına, floresan ROI ortalama normalize yoğunluğunu Ekle &quot;veritabanına spektrum kaydedin.&quot; </li><li> lazer gücü optimize edin ve iyi bir sinyal-gürültü oranı elde edilecek şekilde kazanırlar. Ayarlar farklı ekipman için değişecektir. Arka plan referans olarak floresan olmayan, transfekte edilmemiş hücreler kullanılarak, fakat detektörü (doyma noktasına) dinamik aralığının dışında, hücrelerin parlak olana kadar kazanç ve gücünü artırmak. Arka plan hücre ortalaması sonra saptanabilir flüoresans olmamalıdır. </li><li> aşağıdaki istisnalarla Venus-transfekte hücreler için tekrarlayın: <ol><li> uyarı frekansı 515 nm&#39;de olduğundan emin olun ve bant geçiş filtresi ce olduğuvenus-ifade eden hücreleri tespit ederken 530 nm&#39;ye yakın ntered. </li><li> Olarak &quot;Spektral Mod&quot; 515 nm yerine 458 nm&#39;lik bir eksitasyon frekans kullanımı. </li></ol></li></ol> 5. Yakalama Karışmamış Görüntüler <ol><li> için yakalama modunu değiştirin &quot;Spektral unmixing.&quot; </li><li> Karışmama kanallarına (Şekil 3, Ok-2) venus ve mTFP1 spektral parmak izi ekle. </li><li> geri 458 nm argon kaynağına uyarım lazer ayarlayın. </li><li> 60X petrol amacı yukarıda Nesprin-TS inceleyen tabağına yerleştirin. </li><li> yeteri kadar parlak ifadesi ile bir floresan hücre odaklanan sonra, sinyal-gürültü oranını optimize etmekte kazanç ve lazer gücü ayarlar. NOT: Nesprin-TS FRET verimliliği% 20 yakın olduğu için, karışmamış Venus görüntü karıştırılmamış mTFP1 resimden daha büyük ölçüde sönük olmalıdır. Kabul edilebilir bir güç ve kazanç ayarı iteratif belirlendikten sonra, bu parametreler tüm resimler capt için sabit kalmalıdıredilsin. </li><li> (Tüm doymuş piksel görüntü işleme sırasında çıkartılmıştır) nispeten benzer parlaklık Nesprin-TH hücrelerinin 15-20 görüntülerin en az çekme ve aşırı piksel doygunluk kaçının. </li><li> özdeş görüntüleme parametreleri kullanılarak Nesprin-HL hücreleri ile görüntü yakalama işlemi tekrarlayın. </li></ol> 6. Görüntü İşleme ve Oran Görüntü Analizi <ol><li> Kullanılması açık kaynak ImageJ (FİJİ) yazılımı (http://fiji.sc/), BioFormats Reader kullanılarak açık yerel biçim görüntüler. </li><li> Ön-işlem ve görüntüler ve daha önce tespit edilmiş protokollerin 15 kullanılarak oranı görüntü hesaplamak. </li></ol>

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The authors have nothing to disclose.

Bir yöntem ve Nesprin-2G, nükleer LINC kompleksi içinde bir protein karşısında mekanik gerilimin canlı hücre görüntüleme gösteri, yukarıda özetlenen edildi. Bundan önce işe, örneğin mikropipet aspirasyon sitometrisi manyetik boncuk ve mikroskopik lazer ablasyon gibi çeşitli teknikler, hücre çekirdeği üzerindeki yükü uygulamak ve dökme malzeme özellikleri 16, 17, 18 ölçmek için kullanılmıştır. ...

Kuvvet-duyarlı, genetik olarak kodlanmış FRET sensörler, yakın, canlı hücrelerde çekme bazlı kuvvetleri olarak proteinler, 1, 2, 3, 4 arasında nasıl uygulandığı mekanik kuvvetlerin fikir sağlamak için önemli bir araç olarak ortaya çıkmıştır. Bu araçları ile, araştırmacılar geleneksel floresan mikroskop kullanılarak canlı hücreler içinde, non-invaziv görüntü hücre içi kuvvetler can. Bu sensörler bir elastik peptid 3 ile ayrılmış bir FRET çiftinin (donör ve akseptör floresan proteinleri, en çok mavi bir verici ve sarı akseptör) oluşur. C- veya N-terminal etiketleme aksine, bu sensor, bir molekül gerilme göstergesi gibi davranmak, protein üzerinden iletilen mekanik kuvvetini ölçmek için bir proteinin bir iç alanı içine eklenir. FRET p arasında artan bir mesafe sensörü sonuçları arasında mekanik gerilim artışıhava, azalmış FRET 3 elde edilir. Bunun bir sonucu olarak, FRET ters gerilme kuvveti ile ilgilidir. Bu flüoresan bazlı sensörler fokal yapışma proteinleri (vinkülin 3 ve talin 4), sitoskeletal proteinler (α-aktinin 5), ve hücre-hücre birleşme proteinleri için geliştirilmiştir (E-kadherin 6, 7, VE-cadherin 8 ve PECAM 8). Bu biyoalgılayıcılarda da en sık kullanılan ve iyi karakterize edilmiş esnek bağlayıcı TSmod olarak bilinir ve örümcek ipeği proteini flagelliform türetilmiş 40 amino asitlik (GPGGA) 8, bir tekrarlanan dizinin oluşur. TSmod çekme kuvveti 3 5 pN için 1 FRET yanıtı, bir doğrusal elastik nano yay olarak hareket ettiği gösterilmiştir. flagelliform farklı uzunlukları dinamik r değiştirmek için kullanılabilirTSmod FRET kuvvet duyarlılık 9 ange. Flagelliform ek olarak, spektrin (HP-35 olarak da bilinir) 5 ve villin başlık peptid 4 içindeki kuvvet biyosensörler 4 FRET çifti arasındaki elastik peptidler olarak kullanılmıştır tekrarlar. Son olarak, son bir yay TSmod da sıkıştırma kuvvetlerini 10 algılamak için kullanılabileceğini gösterdi. Son zamanlarda TSmod endojen benzer şekilde davranır mini Nesprin2G (Şekil 2C), olarak bilinen, daha önce geliştirilmiş kesik Nesprin2G protein içine sokulur kullanılarak Nucleo-hücre iskeletinin (LINC) kompleks protein Nesprin2G bağlayıcısına için bir güç sensörü gelişmiş Nesprin-2G 11. LINC kompleksi, nükleer lamina sitoplazmik hücre iskeleti bağlama, çekirdeğin içine dışarıdan yol açan birçok proteinler içerir. Nesprin-2G yapısal bir protein bağlanma her ikisitoplazmada ve çekirdek çevresi alanı GÜNEŞ proteinlerine aktin hücre iskeletinin. Bizim biyosensör kullanarak, Nesprin-2G NIH3T3 fibroblastların 2&#39;de actomyosin bağımlı gerilim tabi olduğunu göstermek başardık. Bu, o kuvvet direkt olarak nükleer LINC kompleksi içinde bir protein karşısında ölçüldü ilk kez oldu ve mechanobiology içinde çekirdeği üzerinde gücünün rolünü anlamak için önemli bir araç haline muhtemeldir. Aşağıdaki protokol, Nesprin- ifade eden hücrelerin FRET görüntülerin memeli hücrelerinde Nesprin gerilim sensörü (Nesprin-TS) ifadesi, hem de toplama ve analizi de dahil olmak, Nesprin-2G güç sensörü nasıl kullanılacağını detaylı bir yöntem sağlar TS. bir spektral detektörü ile donatılmış bir ters konfokal mikroskop kullanılarak, duyarlı emisyon ölçmek için nasıl bir açıklama spektral Karışmama kullanılarak FRET ve rasyometrik FRET görüntüleme sağlanır. çıkış oran ölçer görüntüleri göreceli nereye yapmak için kullanılabilirntitative kuvvet karşılaştırmaları. Bu protokol, fibroblastlarda Nesprin-TS ekspresyonu odaklanmış olsa da, bu hücre hatları ve primer hücreler de dahil olmak diğer memeli hücreleri, kolayca adapte edilebilir. Bundan başka, bu görüntü elde etme ve FRET analizi ile ilgili olarak, bu protokol, hali hazırda diğer proteinler için geliştirilmiştir, diğer FRET&#39;e dayalı bir kuvvet biyosensör için adapte edilebilir.

<table border="0" cellpadding="0" cellspacing="0" width="1183">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-TS DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68127</td>
			<td style="width:475px;">Retrieve from https://www.addgene.org/68127/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-HL DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68128</td>
			<td>Retrieve from https://www.addgene.org/68128/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mTFP1 DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">54613</td>
			<td>Retrieve from https://www.addgene.org/54613/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mVenus DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">27793</td>
			<td>Retrieve from https://www.addgene.org/27793/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">TSmod DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">26021</td>
			<td>Retrieve from https://www.addgene.org/26021/</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Competent Cells</td>
			<td style="width:108px;">Bioline</td>
			<td style="width:119px;">BIO-85026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Liquid LB Media</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10855001</td>
			<td>https://www.thermofisher.com/order/catalog/product/10855001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Solid LB Bacterial Culture Plates</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Ampicillin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Spectrophotometer</td>
			<td style="width:108px;">Biorad</td>
			<td style="width:119px;">273 BR 07335</td>
			<td>SmartSpec Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">quartz cuvette</td>
			<td style="width:108px;">Biorad</td>
			<td align="right" style="width:119px;">1702504</td>
			<td>Cuvette for SmartSpec Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">&#160;DNA isolation kit</td>
			<td style="width:108px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740412.5</td>
			<td>NucleoBond Xtra Midi Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">6-well cell culture dish</td>
			<td style="width:108px;">Falcon-Corning</td>
			<td align="right" style="width:119px;">353046</td>
			<td>Multiwell 6-well Polystrene Culture Dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Dulbecco&#39;s Modified Eagle Medium, (DMEM) cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td>DMEM(1x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16170-078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">reduced serum cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>Reduced Serum Medium, &#34;optimem&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Lipid Carrier Solution</td>
			<td style="width:108px;">invitrogen</td>
			<td style="width:119px;">11668-019</td>
			<td>Lipid Reagent, &#34;Lipofectamine 2000&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">1.5mL sterile plastic tube</td>
			<td style="width:108px;">Denville</td>
			<td style="width:119px;">c2170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Trypsin</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>0.25% Trypsin-EDTA (1x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">glass-bottom microscope viewing dish</td>
			<td style="width:108px;">In Vitro Scientific</td>
			<td style="width:119px;">D35-20-1.5-N</td>
			<td>35mm Dish with 20mm Bottom Well #1.5 glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Fibronectin</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">33016015</td>
			<td>fibronectin human protein,plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">14190-144</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">15mL sterile centrifuge tube</td>
			<td style="width:108px;">Greiner bio-one</td>
			<td align="right" style="width:119px;">188261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">swinging rotor centrifuge&#160;</td>
			<td style="width:108px;">Thermo electron</td>
			<td style="width:119px;">Centra CL2</td>
			<td>Swinging rotor thermo electron 236</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">cell culture biosafety hood</td>
			<td style="width:108px;">Forma Scientific</td>
			<td align="right" style="width:119px;">1284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;climate controlled cell culture incubator</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">3596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">inverted LED widefield fluorescent microscope</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">EVOS FL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Clear HEPES buffered imaging media</td>
			<td style="width:108px;">Molecular Probes</td>
			<td style="width:119px;">A14291DJ</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Fetal bovine Serum</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:483px;">Temperature Controlled-Inverted confocal w/458 and 515nm laser sources&#160;</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:119px;">&#160;LSM 710-w/spectral META detector</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Outgrowth Media</td>
			<td style="width:108px;">Newengland Biolabs</td>
			<td style="width:119px;">B9020s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">NIH 3T3 Fibroblasts</td>
			<td style="width:108px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

Yukarıdaki protokole ardından DNA plazmidi DNA deposundan alınan edildi ve E. coli hücrelerine transforme edildi. Sensör DNA ifade eden E. coli LB / Amfisilin plakaları seçilir ve bir sıvı LB suyu içinde amplifiye edildi. Vektörlerin amplifikasyonu takiben, DNA plazmidleri standart, ticari olarak temin edilebilir DNA izolasyon kiti kullanılarak Tris-EDTA tampon maddesi içinde saflaştırılmıştır. Bir spektrofotometre kullanılarak, saflaştırılmış D...

Watch this Scientific Journal Video about A Protocol for Using FÃƒÂ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

Förster Rezonans Enerji Transferi (FRET) -force biyosensörlerin kullanılması için Protokol Nükleer LINC Kompleksi karşısında Mekanik Kuvvetleri Ölçmek için

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

a-protocol-for-using-frster-resonance-energy-transfer-fret-force

Research

JoVE Journal

Bioengineering

10.5K Görüntüleme.  Virginia Commonwealth University. A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex. 

Video: A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Biyomühendislik

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

Çözüm Biyolojik Makromoleküllerin Küçük ve Geniş Açı X-Ray Saçılma Çalışmaları

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

Kantitatif SENP1 Proteaz Kinetik Belirlenmesi (Förster rezonans enerji transferi) Analizi FRET

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Doku Mühendisliği için iki eksenli Mekanik Yükleniyor Biyoreaktör tasarımı

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Atomik Kuvvet Mikroskobu kullanarak Yaşam Hücrelerinin Mekanik Özellikleri Ölçme

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Yaşam Maya Hücreleri Mitokondriyal Redoks Devlet ve ATP ölçün oranlı metrik Biyosensörler

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.

The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.

Voksellerden Gönderen Bilgi için: Kompleks Elektron Mikroskopi 3D-Data Bölümlere Göre Pratik Rehber

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Mikro-ayağı ile bir mikroakışkan Aygıt Biyofilm Çıtası Formasyonu Protokolü

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lüminesans Rezonans Enerji memeli hücrelerinde ifade Membran Proteinleri Konformasyonel Değişiklikleri Eğitim transfer

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

Robotik Microcontact Baskı (R-μCP) kullanarak Karmaşık Kültürü Substratlar ve Sıralı nükleofilik Giren Fabricating

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...