Philippa Warren Biz onu türlü yardım için teşekkür etmek istiyorum.

1. Sınır Testi: <ol><li> Temel hazırlık: Poly-D-Lizin gecelik ve steril cam slaytlar Odası slaytlar ile kaplı, deney başlamadan önce hazırlanır. Orta Dulbecco&#39;nun Modifiye Kartal,% 10 fetal buzağı serumu (FCS) ile desteklenmiş Orta (DMEM), ve% 1 Penisilin-Streptomisin-Fungizon (PSF) kullanılarak hazırlanmıştır. Ayrıca kalsiyum / magnezyum ücretsiz Hanks dengeli tuz solüsyonu (HBSS) bir şişe hazırlanmıştır. </li><li> Cam şişe içinde kültür İlköğretim sıçan Schwann hücrelerinin% 0.1 tripsin kullanarak 3 dakika tripsinize. İlköğretim sıçan astrosit kültür,% 0.1 &#39;lik tripsin kullanarak 5 dakika tripsinize. Tripsin DMEM ekleyerek% 10 FCS ve 1% PSF ile desteklenmiş aktif değil. </li><li> Tripsinize Schwann hücreleri ve astrositler, 15 ml şahin tüpler ayrı aktarılır ve 300G 5 dakika santrifüj. </li><li> Schwann hücreleri ve astrositler (hücre yoğunluğu varyasyonları temsilcisi sonuçlar bölümüne bakınız) 2 x 10 6 yoğunlukta yeniden askıya alınır. </li><li> Schwann hücre süspansiyonu 50 mcL damla PDL kaplı oda slayt kuyuların bir ucunda yer alır. Bir cam şeridi düz bir kenar oluşturmak için odanın ortasına doğru açılan smear kullanılmıştır. (Cam kesici kullanarak, dikdörtgen bir cam lamelleri sınır deneyleri için kullanılan odasına slaytlar genişliği uygun olacağını boyuna kadar kesilir) </li><li> Astrosit süspansiyon 50 mcL ikinci bir damla yerleştirilir ve aynı sıra ters sonunda, aralarında küçük bir 0.2mm boşluk ile ilk damla smear paralel düz bir kenar oluşturmak için merkeze doğru bulaşmış oldu. </li><li> Odasına damla 37 inkübatöre yerleştirilir ° C&#39;de 2 saat süreyle içeren slaytlar. </li><li> 2 saat sonra, kültürler bağlı olmayan hücresel enkaz kaldırmak için ve taze orta DMEM /% 10% FCS / 1 PSF eklendi içeren DMEM ile yıkandı. Not: DMEM /% 10 FCS /% 1 PSF forskolin (2μM) ve Sığır hipofiz ekstresi (BPE) (10μg / ml) Schwann hücre proliferasyonu uyaran ile takviye edilebilir. BPE, uzun vadeli Schwann hücre kültürü izin için gerekli olan büyüme faktörleri tedarik edecek ve forskolin proliferasyonunu uyardığı Schwann hücre içindeki siklik adenozin monofosfat düzeylerini artırmaktadır. </li><li> Hücreler 8-10 gün boyunca yaklaşık 37 ° C,% 7 CO 2 inkübatör kültür. Orta 3 günde bir değiştirilmesi gereklidir. Iki hücre cephede sonunda birbirlerine ulaşacak ve iki hücre tipleri arasında keskin bir sınır oluşturacak. Bu aşamada deneyler sınır oluşum katılan faktörler analiz yapılabilir. </li><li> Ko-kültür,% 4 formaldehit ve Schwann hücreleri, ko-kültür hücreleri tanımlamak için anti-P75 antikor ve anti-GFAP antikorları astrositler ile boyanmış olabilir ile tespit edilebilir. P75 antikor astrositler eksikliği Schwann hücreleri üzerinde düşük afinite NGF reseptör tanır. Anti-GFAP antikor astrosit sitoplazmasında lokalize glial firbrillary asidik protein tanır. (Bkz. Şekil 1A temsilcisi sınır) </li></ol> 2. Göç Testi: <ol><li> 4 tane iyi plakalar (15mm kuyu) PDL kaplı gecelik ve astrosit mono tabakaları oluşturmak için hazırlanmıştır. </li><li> İlköğretim astrosit kültür,% 0.1 &#39;lik tripsin kullanarak 5 dakika tripsinize. Tripsin DMEM% 10 FCS ve% 1 PSF ve 300G az 5 dakika santrifüj hücre ile desteklenmiş ekleyerek inaktive olur. (Bakınız Şekil 2) </li><li> Astrositler 1x10 5 hücre / ml tekrar askıya alınır. Bu çözeltinin 1 ml 4 kuyucuğu her PDL kaplamalı eklenir. Tek tabaka tamamen konfluent kadar Astrositler 24-48 saat kültüre. </li><li> Paralel, dairesel PDL kaplı cam lamelleri 50 ml tüp yerleştirilir ve küçük cam parçaları oluşturmak için plastik bir pipet kullanarak ezilmiş astrosit mono tabakaları oluşturmak için, astrosit mono tabakaları Schwann hücre göçü değerlendirmek için. </li><li> Cam parçaları 6 plaka çanağı bir kuyunun içine transfer edilir ve büyüklükteki parçaları uygun bir forseps kullanılarak seçilen ve diğer kuyularda yerleştirilir. 6 kuyucuğu her kuyuda 5 cam parçaları yerleştirilir. </li><li> Cam şişe içinde kültür İlköğretim Schwann hücrelerinin% 0.1 tripsin kullanarak 3 dakika tripsinize. Tripsinize Schwann hücreleri, 15 ml şahin tüp ayrı transfer ve 300 G 5 dakika santrifüj. Schwann hücreleri DMEM/10% FCS / 1% PSF 2x 10 6 hücre / mL yeniden askıya alınır. </li><li> Schwann hücrelerinin süspansiyon 20 mcL damlacıkları, 6 kuyu plakaları ve hücreleri yerleştirilen her 5 lamel parçalarının 37 ° C,% 7 2 saat boyunca CO 2 inkübe üzerine eklenir . </li><li> 2 saat sonra, Schwann hücreleri lamel parçaları eklenir ve DMEM/10% FCS /% 1 PSF (forskolin ve 10μg / mL BPE 2μM içerir parçaları tamamen orta ile kaplıdır, böylece her bir kuyunun eklenir) . </li><li> Cam parçaları tamamen con kadar parçaları içeren 6 kuyucuğu 24-48 saat sonra inkübatöre yerleştirilir.Schwann hücreleri ile bilmektedir. </li><li> Bir kez parçaları Schwann hücreleri ile konfluent, her parça dikkatlice keskin forseps kullanarak kaldırdı ve astrosit tek tabaka üzerinde ters yüz aşağı sonra unattached hücreleri çıkarmak ve HBSS batırılmış. </li><li> Her kuyu sonra 1 mL% FCS / 1% PSF ve Schwann hücreleri 24 - 48 dönem için göç etmeye izin verilir DMEM/10 ile kaplıdır. Bu dönemde büyüme faktörleri gibi deneysel reaktifler, Schwann hücrelerinin göç sayısı ve mesafe üzerindeki etkileri değerlendirmek için eklenebilir. </li><li> Geçiş dönemini takiben, cam parçaları, 30 dakika için% 4 paraformaldehid (PFA) ekleyerek her bir kuyunun dibine sabitlenir. Aşağıdaki fiksasyon, P75 Schwann hücreleri (düşük afinite NGF reseptör tanıma), ardından ABC strepavidin kiti-diaminobenzidin (DAB) kullanarak bunları leke Schwann hücrelerinin ışık mikroskobu altında görselleştirme izin immunostain. </li></ol> 3. Temsilcisi Sonuçlar: Schwann astrosit ortak kültürler, 8-10 gün sonra iki hücre tipleri arasında keskin bir sınır göstermektedir. Bu sınır, Schwann hücre proliferasyonu gelişmiş forskolin ve BPE kültür kullanılırsa daha belirgindir. Hiçbir sınır 10 günlük süre içinde görülürse, sınırları kuruluncaya kadar, kültür zaman daha da artmaktadır. Hücre yoğunluğu oranının değiştirilmesi sınır oluşumunu arttırmak için alternatif bir yol. 03:01 Schwann oranı: hücre süspansiyonları 8,18 hazırlarken astrosit hücre yoğunluğunda kullanılabilir. Schwann hücreleri ve astrositler belirlemek için sınır kurulmuş olduğu bir çizgi çizilebilir astrosit topraklarına göç Schwann hücrelerinin sayısı ve floresan mikroskop altında sayılabilir karışmasının hücre değerlendirmek için, immün ardından. Aşağıda, elde edilen görüntüleri iki örnek sınırları, Schwann hücreleri ve astrositler (Şekil 1A) ve hücrelerin, zayıf tekniği (Şekil 1B) nedeniyle farklı toprakları içine de ayrı ayrı değil bir iyi biçimli segregasyon bir. <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="Şekil 1" /> Şekil 1: Sınır tahlil: Schwann astrosit etkileşim. Schwann hücre proliferasyonu uyarmak için forskolin ve BPE varlığı A) 10 gün co-kültür tahlil, iki hücre tipleri (kırmızı = P75, yeşil = GFAP) arasında keskin bir sınır göstermektedir. B) 10 günlük sınır oluşturduğu bir ortak kültür tahlil ve iki hücre cephede karıştırılır. (Kırmızı = P75, yeşil = GFAP) sınır hazırlanması sırasında birbirlerine sonraki astrosit ve Schwann hücresi damlacıkları yerleştirerek Bu iki damlacıklarının karıştırma nedeniyle oldu. <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="Şekil 2" /> Şekil 2 konfluent astrosit tek tabaka Temsilcisi görüntü Göç testleri yüzey üzerinde başka bir hücre tipi, tek bir hücre tipi hareketi değerlendirmek ve bu nedenle sınır oluşum farklı bir fenomen değerlendirmek. Uygulama mono tabakaları üzerine tersini lamelleri sağlamak için gerekli olan Schwann hücre, keskin forseps cam parçaları ve astrosit mono tabakaları kaplı. Zarar yol açmaz Schwann hücreleri, maksimum mesafe göç ve lamel kenarına göç hücrelerinin sayısı göç değerlendirmek için ışık mikroskobu altında ölçülebilir. Aşağıda astrosit tek tabaka yüzeyinde (Şekil 3) DAB boyama P75 antikor ile boyandı Schwann hücrelerinin görüntü. <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="Şekil 3" /> Şekil 3 ters lamel assay kullanarak Göç tahlil. A) Schwann hücresi (kahverengi, P75 antikor ile boyanmış) lamel kenarından uzak göç. Ok lamel uzak hücre göç yönüne işaret eder.

<ol>
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Yukarıda anlatılan testlerin Schwann astrositler ve astrositik ortamda sınırlı Schwann hücre göçü arasındaki sınır oluşumu dahil çoklu faktörlerin rolünü gösteren çeşitli çalışmalarda kullanılmıştır. Stratejilerinin geliştirilmesi optimize etmek için izin ve Schwann hücre greft transplantasyonu takip entegrasyonu geliştirmek ve bunu yaparken de yenileyici aksonlar sağlayan ana dokusu içine greft arka çıkış kolaylaştırmak olduğu gibi bu olayların altı...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41966-029</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-088</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>PSF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>BPE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13028-014</td>
<td>&nbsp;</td>
<tr>
<td>Forskolin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>344273</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips</td>
<td>&nbsp;</td>
<td>VWR-International</td>
<td>631-0149</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips(rectangle)</td>
<td>&nbsp;</td>
<td>MENZEL-GLASER (Supplied by Thermo Scientifics)</td>
<td>BB022050A1</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nunc-LabTek </td>
<td>177380</td>
<td>&nbsp;</td>
<tr>
<td>4 well plates, 6 well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>&nbsp;</td>
<td>4 well plates 
6 well plates</td>
</tr>
<tr>
<td>rat p75 antibody</td>
<td>&nbsp;</td>
<td>Millipore, Temecula,California</td>
<td>MAB365</td>
<td>&nbsp;</td>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>Z0334</td>
<td>&nbsp;</td>
<tr>
<td>Secondary antibodies (Alexa-conjugated)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004 A-11034</td>
<td>Goat anti mouse 568 
Goat anti Rabbit 488</td>
</tr>
<tr>
<td>Secondary biotinylated </td>
<td>&nbsp;</td>
<td>Vector</td>
<td>&nbsp;</td>
<td>Goat anti mouse</td>
</tr>
<tr>
<td>DAB tablets </td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>D4418</td>
<td>&nbsp;</td>
<tr>
<td>ABC elite kit</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps </td>
<td>&nbsp;</td>
<td>FST</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6168</td>
<td>&nbsp;</td>
<tr>
<td>Fluorosave</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>345789</td>
<td>&nbsp;</td>		</tbody>
		</table>
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analysis of schwann-astrocyte interactions using in vitro assays

Schwann hücreleri, omurilik yaralanması sonrasında onarım stratejileri yaygın olarak kullanılan hücre biridir. Schwann hücreleri, aksonal rejenerasyon salgılayan büyüme faktörleri 1,2 destekleyen ve çimlenme ve büyümeyi teşvik adezyon molekülleri 3 ve ekstraselüler matriks molekülleri 4 sağlayarak yeteneğine sahiptir . Buna ek olarak 5 yaralanma yerinde demyelinated aksonlar myelinate. Ancak, Schwann hücrelerinin nakli aşağıdaki implant sitesinden göç yoktur ve ev sahibi astrositler 6,7 ile karışmaz. Bu sonuç, Schwann hücreleri ve astrosit arasında keskin bir sınır oluşumu proksimale ve distale ana dokusu içine greft arka çıkmak için çalışıyor büyüyen aksonlar için bir engel oluşturmak. Schwann hücreleri ile temas Astrositler da hipertrofisi geçmesi ve inhibitör molekülleri 8-13 up-regüle. In vitro tayinlerde modeli Schwann hücre astrosit etkileşimleri kullanılmış olan ve hücresel davranışın altında yatan mekanizma anlaşılmasında önemli olmuştur. In vitro tayinlerde Bu sınır tayini, bir ko-kültür, sadece küçük bir boşluk iki hücre cephede ayıran farklı toprakları işgal her hücre tipi ile iki farklı hücre kullanılarak yapılır . Hücrelere bölmek ve göç gibi, hücresel iki cephede birbirlerine yakın ve nihayet çarpışır olsun. Bu davranış sınırında analiz edilecek iki hücre popülasyonlarının sağlar. Aynı tekniğin başka bir varyasyonu iki hücre popülasyonları kültür karışımı ve zamanla iki hücre tipleri ayırınız Schwann hücreleri ile birlikte birden fazla Schwann astrosit sınırları astrositler arasındaki adalar olarak bir araya clumped. Iki hücre tipleri etkileşim eğitim kullanılan ikinci tahlil, hücresel hareketi diğer hücre tipi tek tabaka 14,15 yüzey üzerinde takip edilebilir göç testtir . Bu testte ters lamel testinin yaygın olarak bilinir. Schwann hücreleri, küçük cam parçaları üzerinde kültür ve astrosit mono tabakaları yüzey üzerine yüzü aşağı gelecek şekilde ters düz ve göç lamel kenarında değerlendirilir. Her iki deneyleri hücresel dışlanma ve sınır oluşum yer altında yatan mekanizmaları inceleyerek etkili olmuştur. Bazı moleküllerin bu teknikleri N-Cadherins 15, Kondroitin Sülfat proteoglikanların (CSPGs) 16,17, FGF / Heparin 18, Efesliler / Ephrins 19 kullanılarak belirlendi. Bu makale, kademeli moda sınır tahlil ve göç tahlil tanımlamak ve meydana gelebilecek olası teknik sorunları aydınlatmak için niyetindedir.

Watch this Scientific Journal Video about Analysis of Schwann-astrocyte Interactions Using In Vitro Assays at JoVE.com

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Bu makale niyetinde kademeli biçimde tanımlamak için yaygın olarak kullanılan<em&gt; In vitro</emSchwann hücre asrtocyte etkileşimleri inceleyerek kullanılan deneyleri.

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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JoVE Journal

Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.2K Görüntüleme.  University of Cambridge. Bu makale niyetinde kademeli biçimde tanımlamak için yaygın olarak kullanılan In vitro

Video: Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Biyolojik Tahliller Manyetik Modülasyon Biyoalgılayıcı Sistemi kullanarak hızlı Homojen Algılama

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Biyoloji

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ bağımlı CFTR makromoleküler Sinyal Kompleksleri İn Vitro Analizi

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Vitro Hücre Göç ve Invasion Tahliller

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Pre-mRNA Substratlarm 3 &#39;End Bölünme: Hücre Ücretsiz Sistemlerini Kullanma RNA İşleme Reaksiyonları Analizi<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro toplama Deneyler hiperfosforile tau proteini kullanılarak

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Spri, Nano-Spri ELISA Tahliller Kullanma Serum İnsan Büyüme Hormonu Karşılaştırmalı Analizi

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Explanted Retina doku hücre dışı akı analizi ile enerji metabolizmasında ölçümü

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Floresan proteinlerin etkisi Polyglutamine toksisite deneyleri Maya kullanılarak füzyon ortakları

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

İki adımlı PCR ve Yeni Nesil 16S rRNA Gen Sıralaması Kullanarak Mikrobiyota Analizi

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...