İş bizim laboratuar Ulusal Sağlık Enstitüleri hibe tarafından desteklenmektedir.

Hazırlık <ul><li> Lameller otoklava olmalıdır. </li><li> Steril distile veya deiyonize su çözümler hazırlamak ve çamaşır lamelleri için kullanılmalıdır. </li><li> AC (% 40 w / v) ve bis-AC (% 1 w / v) çözümleri 0.2 mikron filtre ile sterilize edilir. Kullanımı ve steril filtre kısa bir süre önce,% 10 amonyum persülfat (100μg/ml su APS) hazırlayın. APS çözüm aylık değiştirin. </li><li> 3-APTMS, kloroform, glutaradehyde, NHS ve SurfaSil gibi kimyasal reaktifler otoklavlanmalıdır sadece hidrojellerin hazırlanması için kullanılan atanmış bir şişe içinde tutulur. </li><li> En iyi sonuç için, hidrojeller uygun ECM protein ile geceleme inkübasyondan sonra birkaç gün içinde kullanılabilir olmalıdır. </li><li> Hidrojel dökülmesinden önce, 50 ml polipropilen Falcon tüp kloroform (10 ml genelde 20 üst lamelleri için yeterli)% 10 SurfaSil çözüm hazırlayın. (Bizim laboratuvar genellikle% 0.5 glutaraldehid kuluçka adımı sırasında üst lamelleri siliconizes). En az 10 dakika süreyle Falcon tüp ve kaya lamelleri ekleyin. SurfaSil çözüm Durusu ve hava hidrojeller hazırlanacaktır biyolojik güvenlik kabini Kimwipes lamelleri kuru. </li><li> Isı ile inaktive edilmiş BSA çözüm aşağıdaki gibi hazırlayın: PBS içinde yağ asidi serbest BSA 20 mg / ml solüsyon 30 dakika süreyle 68 ° C su banyosunda inkübe edilir. Çözüm, daha sonra steril 4 ° C&#39;de filtre ve depolanan </li></ul> Prosedür <ol><li> 150 mm Petri kabında alt yarısında Parafilm bir kat yerleştirin. </li><li> 9 Parafilm üstüne 25 mm lamelleri yukarı bakacak şekilde yerleştirin ve 1 ml 0.1 M NaOH ile onları kapsayacak. 3 dakika sonra bir vakum hattı ile aspire inkübe. </li><li> Her lamel kimyasal bir davlumbaz, 3 yerde 0.5 ml APTMS çalışma. 3 dakika inkübe edin ve sonra APTMS aspirat. Çok uzun süre beklerseniz, bir köpük oluşacaktır. </li><li> Lamelleri aynı çanak 20 ml deiyonize su ile durulayın. Kavisli bir forseps kullanarak çanak lamelleri çıkarın ve onların tedavi tarafı yeni bir 150-mm çanak yukarı bakacak şekilde aktarabilirsiniz. Her bir yıkama için 10 dakika, rocker, lamelleri üç kez deiyonize su ile yıkayın. Tüm APTMS kaldırmak için başarısız olursa, bir sonraki adımda glutaraldehid tepki ve beyaz bulutlu bir çökelti (Şekil 1) bırakın. </li><li> Glutaraldehit Çözülme (~ kullanmadan önce 10 dakika). </li><li> Kavisli bir forseps kullanarak, temiz bir tabak Parafilm katmanlı lamelleri transferi ve kalan herhangi bir sıvı aspire. Gerektiği gibi kalan suyu örtecek bir vakum hattı veya Kimwipe kullanın. </li><li> Her lamel 0.5 ml steril deiyonize su içerisinde% 0.5 glutaraldehid ile tamamen örtün ve 30 dakika kimyasal bir kaput inkübe edin. Glutaraldehid aspire. Durulayın ve 4. adımda lamelleri yıkayın. Lamelleri tamamen kurulayın. [Burada durdurmak ve kuru bir alanda birkaç hafta lamelleri bırakabilirsiniz]. </li><li> Hidrojeller hazırlamak için hazır olduğunuzda, lamelleri, reaktif yüzü yukarı, biyolojik güvenlik kabini yüzeye bantlanmış olmuştur Parafilm bir sayfa aktarmak için kavisli bir forseps kullanın. Lamelleri Parafilm yüzey üzerinde düz olduğundan emin olun. </li><li> Toluen doymuş bir NHS çözüm hazırlayın. (Özellikle deney için yeterli toluen NHS küçük bir miktar çözülür. NHS artık eriyene kadar biraz NHS biraz ekleyerek tutun. Doymuş çözüm genellikle bulutlu ve pembe.) </li><li> Daha sonra, istenen akrilamid yüzde ulaşmak için akrilamid, bis-akrilamid, su ve APS hazırlar. 0.8ml toplam hacmi aşağıda özetlenen mikrosantrifüj tüpler reaktifler ekleyin. </li><li> Daha sonra, her seferinde bir kısım, NHS ekleyin ve 0.8 ml AC çözüm TEMED, girdap kısa ve DERHAL biyolojik güvenlik kabini lamel başına 140 ul kullanarak 3-5 jeller dökün. Mümkün olduğunda, bu noktadan itibaren tüm adımları bir biyolojik güvenlik kabini yapılmalıdır. [Not: ihtiyaca göre farklı boyut lamelleri göre kullanılabilir. 18 mm lamelleri kullanılması durumunda, lamel ortalama ~ 33 ul AC çözümü ve 18 mm üst silikonlu lamel kullanın.] <table border="1"><tbody><tr><td></td><td colspan="4"> Bis-AC (%) </td></tr><tr><td></td><td> 0.3 (sert) </td><td> 0,15 </td><td> 0,06 </td><td> 0.03 (yumuşak) </td></tr><tr><td></td><td> ul </td><td> ul </td><td> ul </td><td> ul </td></tr><tr><td> Su </td><td> 402 </td><td> 522 </td><td> 594 </td><td> 618 </td></tr><tr><td> AC </td><td> 150 </td><td> 150 </td><td> 150 </td><td> 150 </td></tr><tr><td> Bis-AC </td><td> 240 </td><td> 120 </td><td> 48 </td><td> 24 </td></tr><tr><td> APS </td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td></tr><tr><td> TEMED</td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> NHS </td><td> 228 </td><td> 228 </td><td> 228 </td><td> 228 </td></tr></tbody></table></li><li> Polimerize başlamadan önce her jel üstünde silikonlu 25 mm lamel hızla yerleştirin. Üst lamel eklenmesi AC tamamen alt lamel karşılamak için izin vermelidir. Bu &quot;sandviç&quot; AC polimerize kadar oda sıcaklığında inkübe edin. (Polimerizasyon oluştu belirlemek için mikrosantrifüj tüp içinde kalan AC çözüm kontrol edin. Genellikle birkaç dakika sert jeller ve yumuşak olanlar için biraz daha uzun yeterli olacaktır.) </li><li> Dikkatle sandviç pick up (steril eldiven) ve çıkıntılar polimerize jel kadar üst lamel kapalı slayt. Daha sonra jel kapalı gözetlemek. Üst lamel çıkarmadan önce çok uzun süre beklemek durumunda lamel kaldırılmış olduğundan, jel rip. </li><li> Üst lamel atın. 2 ml PBS / alt jel lamelleri (bundan sonra hidrojel denir) 6-iyi tabak içine yerleştirin. PBS, her kuyuya jel-lamel önce veya sonra eklenebilir. Rocker, yıkama başına 5 dakika hidrojeller, üç kez PBS ile yıkayın. </li><li> Hidrojellerin gerekli sayıda hazırlanmıştır kadar tekrarlayın 11-14 adımları. </li><li> Fibronektin solüsyonu (PBS içinde 3 mg / ml) veya diğer ECM protein (Klein ve ark. Ayrıntılar için 13) 2 ml her hidrojel örtün. ECM protein kovalent 4 ° C&#39;de gece inkübasyon sırasında hidrojel bağlı olur </li><li> , En az 30 dakika süreyle 37 ° ECM çözümü ve serum ücretsiz medya 1mg/ml ısı ile inaktive edilmiş yağ asidi serbest BSA blok girmemiş NHS aspire ° C hücre kültürü inkübatörde. Hidrojeller steril PBS veya hücre kültür ortamı ile bir kez durulayın. </li><li> FBS içeren uygun bir kültür ortamı Levha hücreleri. Hidrojel numaralı seribaşı hücrelerin sayısı, hücre deneyleri için gerekli yayılması ve confluency derecesi dayalı kullanıcı tarafından belirlenmelidir. Yaklaşık 10 5 hücreleri genellikle batı ve qPCR blot analizi için yeterli olacaktır. </li><li> Kuluçka döneminin ardından, hücrenin protein ya da mRNA elde edilebilir. Lamelleri kuyulardan kavisli bir forseps kullanarak dikkatlice çıkarılır ve laboratuarlarında Parafilm bir sayfa boyunca (2-3cm) aralıklı edilmiştir lizis tamponu 100 ul damlacıkları üst (hücre tarafı aşağı) yerleştirilir. (Biz batı blot örnekleri hazırlamak ve qPCR için RNA izole etmek için, sırasıyla standart SDS örnek tampon ve TRIzol kullanın.) Lizis tamponu ile hücrelerin tam bir dakika boyunca inkübe edin. Lamelleri çıkarın ve bir mikrosantrifüj tüp lizis tamponu aktarmak. Alternatif olarak, kullanıcı sadece RNA çıkarılması için yeni bir 6-plaka Her hidrojel transferi ve 1 ml TRIzol / iyi ekleyebilirsiniz. 3 dakika boyunca inkübe ve mikrosantrifüj tüp depolama için TRIzol çözüm kaldırmak. </li></ol> Immünofloresan olarak prosedürler hakkında ek bilgi, hidrojeller üzerinde seribaşı hücreler için BrdU boyama, transfeksiyon, vb Klein ve ark. 13, 2007. Temsilcisi Sonuçlar APTMS ek lamelleri iyice yıkama &quot;reaktif&quot; lamelleri üretiminde önemli bir adımdır. APTMS tamamen kaldırmak için başarısız olursa, sonraki adımda glutaraldehid tepki ve Şekil 1A görüldüğü gibi beyaz bulutlu çökelti üretmek. Şekil 1B, uygun bir şekilde yıkanır ve kurutulur lamel gösterir. Çökelti oluşursa lamel artık deresindeki tüm prosedürü başından itibaren yeniden başlatılması gerekir. Gecede ECM proteinleri ile hidrojel oluşumu ve kaplama sonrasında, hücrelerin ertesi gün seribaşı olabilir. Şekil 2&#39;de görüldüğü gibi, sert karşı yumuşak hidrojeller yayılan hücre arasında belirgin bir fark vardır. Fare embriyonik fibroblastlar (MEFS) Phalloidin boyama tarafından görüleceği gibi yumuşak hidrojeller ile karşılaştırıldığında, hücre sert büyük ölçüde yayılmıştır. Gerçekten de, en hücreleri yumuşak hidrojel bağlama kompakt kalacaktır ve daha verimli bir şekilde takın. Hücre yayılmasında fark sadece MEF morfolojisi Şekil 2&#39;de gösterildiği gibi olmasına rağmen, diğer birçok hücre hatları tutarlı 11-12,14 test. Başarı sırları <ol><li> Işlemi başladıktan sonra, &quot;reaktif&quot; yan lamelleri tutmak ve hangi tarafı kaplanmış akılda tutmak çok önemlidir. </li><li> Eldivenler işlem sırasında mümkün olduğu kadar steril bir çalışma ortamı sağlamak için tüm zamanların giyilmelidir. </li><li> Ilk adımlar çoğu kimyasal bir davlumbaz ve laboratuar tezgah arasında yapılmaktadır. Lamelleri fazla glutaraldehid çıkarmadan sonraki tüm adımlar, biyolojik güvenlik kabini altında yürütülmelidir. </li><li> Hücre sprea farklılıklar nedeniyleding (Şekil 2) sert hidrojeller ile karşılaştırıldığında, yumuşak hidrojeller hücre sayısının iki katını ekim tavsiye ederiz. </li><li> AC polimerizasyon işlemi son derece hızlı. Ilk kez kullananlar için, bir APS miktarını azaltabilir veya polimerizasyon süreci uzatmak için çözüm TEMED. Bir defada birkaç lamelleri daha hazırlamak için kalkmayın. </li><li> G0 senkronizasyon gereklidir hücre döngüsü çalışmalarda, genellikle plastik kültür kaplarına hücreler serum açlıktan hücreleri trypsinize ve mitojenler varlığı (FBS ve / veya büyüme faktörleri) farklı uyumluluklar hidrojeller onları reseed. Bu prosedür G0 senkronize hücrelerinin başlangıç ​​nüfus tüm örneklerde aynı olmasını sağlar. Ancak, birçok sinyal iletimi çalışmaları için gerekli mitojenle stimülasyon süresi eki ve hücrelerin yayılmasını sağlamak için yeterince uzun olmayabilir. Bu durumda, biz hidrojeller hücreler serum açlıktan ve sonra doğrudan mitojen ile onları uyarmak. </li></ol><img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="Şekil 1a" /> Şekil 1A. Yanlış lamel yıkanmış APTMS ek ardından, lamel glutaraldehid çözüm ek önce 1-2 dakika yıkandı. Usul özetlenen adımları göre yıkanmamış lamel bir çökelti oluşturur. <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="Şekil 1b" /> Şekil 1B. Düzgün lamel yıkanır. APTMS sonra eklenmesi, lamel glutaraldehid çözümünün yanı sıra her biri 10 dakika önce üç kez yıkandı . Lamel çökelti oluşturur. <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="Şekil 2" /> Şekil 2. Sertlik farklı. Hidrojeller hücre Morfoloji MEFS 9 saat boyunca yüksek veya düşük sertlik fibronektin kaplı hidrojeller numaralı seribaşı kuruldu. Kuluçka döneminin ardından, hücreler, sabit permeabilize ve f-aktin bağlanır FITC-Phalloidin ile boyandı. Yüksek sertlik jeller MEFS düşük sertlik hidrojeller numaralı seribaşı göre stres liflerin sergilemek ve iyi yayılır. Ölçek çubuğu = 50μm. <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="Şekil 3" /> Şekil 3. Temsilcisi fare siklin D1 mRNA düzeyleri kantitatif PCR sonucu serum açlıktan fare embriyonik fibroblastlar yüksek (% 3 akrilamid) veya düşük (% 0.3 akrilamid) sertlik hidrojeller üzerine kaplama ve 9 saat 10% FBS ile uyarılır. RNA ekstraksiyonu takiben siklin D1 mRNA düzeyleri (18S RNA normalize), kantitatif gerçek zamanlı PCR analizi yapıldı. G0 siklin D1 mRNA latent hücreleri temsil eder. Siklin D1 mRNA düzeyleri sert hidrojeller değil yumuşak hidrojeller önemli ölçüde artırır. SD yinelenen PCR reaksiyonları - Veri + / ortalama.

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Hidrojel polimerizasyon işlemi çok önemli bir unsur hücreleri ECM-kaplı hidrojel kendini daha ziyade cam lamel bağlamak sağlayacak hava kabarcığı oluşumunu önlemek için. Bu vorteks ve görsel hiçbir hava kabarcığı jel tuzak haline emin sonra dikkatli bir şekilde polimerizasyon çözüm pipetleme önlenebilir. Biz her zaman deneyler için yeterince sağlamak için ek &quot;reaktif&quot; lamelleri ve hidrojellerin hazırlanması önerilir. Jeller PBS ile yıkanır zaman özel...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

Doku sertliği hücresel fonksiyonunun önemli bir belirleyicisi olup, doku sertliği değişiklikler yaygın fibrozis, kanser ve kardiyovasküler hastalık 1-11 ile ilişkilidir. Geleneksel hücre biyolojik yaklaşımlar hücresel fonksiyonu çalışmalarında elastik bir ECM ya da dokular arasında ECM sertlik değişikliklerin etkisini dikkate almaz, katı bir alt tabaka üzerinde kültür hücreleri (plastik tabaklar ya da cam lamelleri) içerir. In vitro, in vivo doku uyumu şartları model için, biz ve diğerleri ECM-kaplı hidrojeller kullanın. Laboratuvarımızda, hidrojeller dizi 12 biyolojik olarak görülen doku uygunluk bildirimleri taklit edebilir poliakrilamid dayanmaktadır. &quot;Reaktif&quot; kapak fişleri 3-APTMS Ayrıca NaOH ile kuluçka tarafından üretilir. Glutaraldehyde 3-APTMS ve poliakrilamid jel çapraz bağlamak için kullanılır. Akrilamid (AC), bis-akrilamid (Bis-AC) ve amonyum persülfat bir çözüm hidrojel polimerizasyon için kullanılır. N-hydroxysuccinimide (NHS), hidrojel çapraz ECM protein AC çözüm içine dahil edilmiştir. Hidrojel polimerizasyonu sonra jel yüzeyi, fibronektin, vitronectin, kollajen, vb gibi bir seçim ECM protein ile kaplıdır Bir hidrojel sertlik reoloji veya atomik kuvvet mikroskobu (AFM) tarafından belirlenir ve çözüm 12 AC ve / veya bis-AC yüzdesi değişen ayarlanabilir. Bu şekilde, alt tabaka sertlik reoloji veya AFM kullanılarak belirlenebilir Biyolojik dokuların sertlik eşleştirilmiş olabilir. Hidrojeller ve kültürlü gerekli deneysel koşullar dayalı bu hücreler daha sonra numaralı seribaşı olabilir. Moleküler analiz için hücre ve onların kurtarma Görüntüleme basittir. Bu yazı için, yumuşak katmanlarından 20.000 Pascal &lt;E 3000 Pascal ve sert doku katmanlarından /&gt; elastik modülü (E) olarak tanımlar.

Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Hücresel fonksiyonları katmanlarından sertlik etkisi modellenmiş olabilir<em&gt; In vitro</em&gt; Uyumluluklar değişen poliakrilamid hidrojeller.

Matrix Rijitlik Akrilamid tabanlı Hidrojeller kullanarak Hücresel Fonksiyon Üzerindeki Etkilerinin incelenmesi

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

studying-effects-matrix-stiffness-on-cellular-function-using

Research

JoVE Journal

Biology

21.9K Görüntüleme.  University of Pennsylvania. Hücresel fonksiyonları katmanlarından sertlik etkisi modellenmiş olabilir In vitro Uyumluluklar değişen poliakrilamid hidrojeller.

Video: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Mitoz incelenmesi için Mikroenjeksiyon Teknikleri Drosophila melanogaster Sinsityal Embriyo

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Biyoloji

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Doku Mühendisliği 3D Hidrojeller Hücresel Kapsülleme

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Optik Tuzak kullanarak Bakteriyel Hücre Bükme Rijitlik Ölçüm

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Endojen Wild-tip BRCA1 Express hücreler kullanarak Homolog Rekombinasyon BRCA1 Mutasyonlar Etkileri belirlenmesi

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Polarize Epitel Hücreleri içine Plazmidler, Mikroenjeksiyon Küçük GTPases Fonksiyon Analizi

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Sitokrom kullanarak Mitokondriyal Solunum Fonksiyon Görselleştirme C Oksidaz / Succinat Dehidrogenaz (COX / SDH) Çift-etiketleme Histokimya

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Üzerine Hipoksi Etkileri Eğitim için tanımlı Gazlı ortamlar oluşturma<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Zebra balığı adacıkları tek hücreli çözünürlük kalsiyum Imaging'i kullanma Beta hücre fonksiyonu Analizi

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Protein ve Mikro-RNA Modülasyonunun Adipozit İşlevi Üzerindeki Etkisini Incelemek için Bir Adiposit Hücre Kültürü Modeli

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In Vitro E3 Ubiquitin Ligase fonksiyonunun analizi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...