Gostaríamos de agradecer a Filipa Warren por sua ajuda espécie.

1. Ensaio de fronteira: <ol><li> Preparação básica: slides Câmara são revestidas com poli-D-lisina lâminas de vidro durante a noite e estéreis são preparados antes de iniciar o experimento. Médio está preparado usando Médio Modified Dulbecco Águia (DMEM), suplementado com 10% de soro fetal bovino (FCS), e 1% penicilina-estreptomicina Fungizon (PSF). Também uma garrafa de cálcio / magnésio livre solução salina equilibrada Hanks (HBSS) é preparado. </li><li> Células de Schwann de rato primários cultivados em frasco são tripsinizados por 3 minutos utilizando Tripsina 0,1%. Cultura primária de astrócitos de ratos é tripsinizados por 5 minutos, utilizando tripsina 0,1%. Tripsina é inativada pela adição de DMEM suplementado com 10% SFB e PSF 1%. </li><li> Células de Schwann e astrócitos tripsinizados são transferidos para tubos de 15 mL em separado falcon e centrifugado a 300G por 5 minutos. </li><li> Células de Schwann e astrócitos são re-suspenso na densidade de 2 x 10 6 (ver secção representativa resultados para variações de densidade celular). </li><li> 50 mL queda de Schwann suspensão de células é colocado em uma extremidade do PDL poços deslize revestido de câmara. A faixa de vidro foi usado para manchar a queda em direção ao centro da câmara para gerar uma borda em linha reta. (Usando um cortador de vidro, um lamínulas de vidro retangular são cortadas longitudinalmente de modo que caberia a largura da câmara de slides utilizado para ensaios de limite) </li><li> A segunda queda de 50 mL de suspensão de astrócitos foi colocado no extremo oposto do mesmo poço e manchada em direção ao centro para criar uma borda em linha reta paralela ao esfregaço primeira queda com um pequeno intervalo 0,2 milímetros entre eles. </li><li> A câmara de slides contendo as gotas são colocadas na estufa a 37 ° C por 2 horas. </li><li> Após 2 horas, as culturas foram lavadas com DMEM para remover não-inscritos restos celulares e meio fresco contendo DMEM / 10% FCS / 1% de PSF acrescentou. Nota: DMEM / 10% FCS / 1% de PSF podem ser complementados com forskolin (2μM) e extrato de hipófise bovina (BPE) (10μg / mL) estimulam a proliferação das células de Schwann. BPE vai fornecer os fatores de crescimento necessário para permitir cultura de células de Schwann e de longo prazo forskolin aumenta o monofosfato de adenosina cíclico níveis dentro das células de Schwann para induzir a proliferação </li><li> As células são cultivadas por cerca de 80-10 dias a 37 ° C, 7% incubadora de CO 2. O meio precisa ser trocada a cada 3 dias. As duas frentes de células acabará por alcançar um ao outro e uma fronteira nítida irá formar entre os dois tipos de células. Nesta fase os experimentos podem ser realizados análise dos fatores envolvidos na formação de fronteira. </li><li> Co-cultura pode ser fixado com formol 4% e as células de Schwann podem ser imunocoloração com anticorpo anti-P75 e astrócitos com anti-GFAP anticorpos para identificar as células em co-cultura. P75 anticorpo reconhece receptor de baixa afinidade NGF em células de Schwann que astrócitos falta. Anti-GFAP anticorpo reconhece a proteína glial ácida firbrillary que localiza-se citoplasma de astrócitos. (Por favor, veja Figura 1A de fronteira representante) </li></ol> 2. Ensaio de migração: <ol><li> 4 pratos bem (15mm poços) são PDL revestido durante a noite e preparados para a criação de monocamadas de astrócitos. </li><li> Cultura de astrócitos primário é tripsinizados por 5 minutos, utilizando tripsina 0,1%. Tripsina é inativada pela adição de DMEM suplementado com 10% SFB e PSF 1% e de células centrifugadas a 300G por 5 minutos. (Veja Figura 2) </li><li> Astrócitos são re-suspenso em 1x10 5 células / mL. 1 mL desta solução é adicionada a cada revestido PDL bem em 4 pratos bem. Astrócitos são cultivadas por 24-48 horas até que a monocamada é completamente confluentes. </li><li> Para avaliar a migração das células de Schwann em monocamadas de astrócitos, em paralelo com a criação de monocamadas de astrócitos, circular lamínulas PDL-revestido de vidro são colocados em um tubo de 50 mL e esmagado com uma pipeta de plástico para criar pequenos fragmentos de vidro. </li><li> Fragmentos de vidro são transferidos para um poço em 6 prato prato bem e usando uma pinça adequada fragmentos de tamanho são escolhidos e colocados em outros poços. 5 fragmentos de vidro são colocados em cada poço em 6 pratos bem. </li><li> Pilhas de Schwann cultivadas em frasco são tripsinizados por 3 minutos utilizando tripsina 0,1%. Células de Schwann tripsinizados são transferidos para separar 15 mL tubo falcon e centrifugado a 300 G durante 5 minutos. Células de Schwann são re-suspenso em 2x 10 6 células / mL em DMEM/10% PSF FCS / 1%. </li><li> 20 gotas mL da suspensão de células de Schwann é adicionado em cada cinco fragmentos de lamela colocada em 6 placas bem e as células são incubadas a 37 ° CO C, 7% 2 por 2 horas. </li><li> Após 2 horas, as células de Schwann são presos aos fragmentos lamela e DMEM/10% FCS / 1% de PSF (complementado com 2μM de forskolin e 10μg / mL da BPE é adicionado a cada poço, para que os fragmentos são completamente cobertos com o meio) . </li><li> Os 6 pratos bem contendo os fragmentos são então colocados na incubadora por 24-48 horas até que os fragmentos de vidro são totalmente confluente com células de Schwann. </li><li> Uma vez que os fragmentos são confluentes com células de Schwann, cada fragmento é pego com cuidado usando uma pinça afiada e mergulhadas em HBSS para remover as células solto e então veremos face invertida para baixo sobre a monocamada de astrócitos. </li><li> Cada poço é então coberto com 1 mL de DMEM/10% de células FCS / 1% de PSF e Schwann têm permissão para migrar para o período 24-48. Durante este período experimental reagentes, tais como fatores de crescimento podem ser adicionados para avaliar seu efeito sobre o número ea distância de migração de células de Schwann. </li><li> Após o período de migração, os fragmentos de vidro são fixos ao fundo de cada poço, adicionando paraformaldeído a 4% (PFA) por 30 minutos. Fixação seguintes, as células de Schwann immunostain para p75 (reconhecendo baixa afinidade do receptor NGF), em seguida, mancha-los usando ABC strepavidin kit-diaminobenzidina (DAB), para permitir a visualização das células de Schwann em microscópio de luz. </li></ol> 3. Resultados representativos: Após 8-10 dias Schwann-astrócito co-culturas mostram uma fronteira nítida entre os dois tipos de células. Este limite é mais pronunciada se forskolin e BPE é utilizado na cultura como a proliferação das células de Schwann é reforçada. Se nenhum limite é observado durante o período de 10 dias, o tempo da cultura pode ser aumentada até que os limites são estabelecidos. Mudando a relação entre a densidade celular é uma forma alternativa para aumentar a formação de fronteira. A proporção de 3:1 Schwann: densidade de células astrócitos podem ser usados ​​na preparação de suspensões de células 8,18. Seguintes imunocoloração para identificar as células de Schwann e astrócitos, células para avaliar mistura uma linha pode ser desenhada onde o limite é estabelecido eo número de células de Schwann migrando para o território astrócitos podem ser contados em microscópio de fluorescência. Abaixo estão dois exemplos de imagens obtidas fronteiras, com uma bem formada a segregação de células de Schwann e astrócitos (Figura 1A) e aquele em que as células não têm segregado bem em diferentes territórios, devido à má técnica (Figura 1B). <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="Figura 1" /> Figura ensaio Limite 1:. Schwann-astrócito interação. A) A 10 dias do ensaio de co-cultura na presença de forskolin e BPE para estimular a proliferação das células de Schwann demonstra uma fronteira nítida entre os dois tipos de células (vermelho = P75, verde = GFAP). B) Um dia 10 ensaio de co-cultura em que a fronteira não está formado e as duas frentes de células mistas. Isso ocorreu devido à mistura das duas gotas ao colocar gotas de células de Schwann e astrócitos ao lado do outro durante a preparação do limite (vermelho = P75, verde = GFAP). <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="Figura 2" /> Figura 2. Imagem representativa de monocamada confluente astrócitos Ensaios de migração avaliar o movimento de um tipo de célula sobre a superfície de outro tipo de célula e, portanto, avaliar um fenômeno diferente da formação de fronteira. Prática é necessária para garantir invertendo as lamelas em monocamadas não levar a danos à célula de Schwann cobertas de fragmentos de vidro e monocamadas de astrócitos pelo fórceps afiada. Para avaliar a migração de células de Schwann, a distância máxima da migração e do número de células migram a partir da borda da lamínula pode ser medido em microscópio de luz. Veja abaixo a imagem de células de Schwann corados com anticorpo P75 usando coloração DAB na superfície da monocamada de astrócitos (Figura 3). <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="Figura 3" /> Figura ensaio de migração 3. Lamela usando ensaio invertido. A) As células de Schwann (marrom, imunocoloração com anticorpo p75), que migram para longe da borda da lamínula. Seta aponta para a direção da migração de células de distância da lamínula.

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+transplanted+into+normal+and+X-irradiated+adult+white+matter+do+not+migrate+extensively+and+show+poor+long-term+survival.">Schwann cells transplanted into normal and X-irradiated adult white matter do not migrate extensively and show poor long-term survival.</a> Exp Neurol. 164, 292-302 (2000).</li><li>Lakatos, A., Franklin, R. J., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+and+Schwann+cells+differ+in+their+in+vitro+interactions+with+astrocytes.">Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.</a> Glia. 32, 214-225 (2000).</li><li>Lakatos, A., Barnett, S. C., Franklin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+induce+less+host+astrocyte+response+and+chondroitin+sulphate+proteoglycan+expression+than+Schwann+cells+following+transplantation+into+adult+CNS+white+matter.">Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white matter.</a> Exp Neurol. 184, 237-246 (2003).</li><li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Schwann+cell+interactions+in+culture.">Astrocyte-Schwann cell interactions in culture.</a> Glia. 11, 367-377 (1994).</li><li>Ghirnikar, R. S., Eng, L. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrogliosis+limits+the+integration+of+peripheral+nerve+grafts+into+the+spinal+cord.">Astrogliosis limits the integration of peripheral nerve grafts into the spinal cord.</a> Brain Res. 277, 175-180 (1983).</li><li>Fok-Seang, J., Mathews, G. A., ffrench-Constant, C., Trotter, J., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+oligodendrocyte+precursors+on+astrocytes+and+meningeal+cells.">Migration of oligodendrocyte precursors on astrocytes and meningeal cells.</a> Dev Biol. 171, 1-15 (1995).</li><li>Wilby, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+migration+is+integrin-dependent+and+inhibited+by+astrocyte-produced.">Schwann cell migration is integrin-dependent and inhibited by astrocyte-produced.</a> Glia. , (2010).</li><li>Santos-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF/heparin+differentially+regulates+Schwann+cell+and+olfactory+ensheathing+cell+interactions+with+astrocytes:+a+role+in+astrocytosis.">FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.</a> J Neurosci. 27, 7154-7167 (2007).</li><li>Afshari, F. T., Kwok, J. C., Fawcett, J. 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Os ensaios descritos acima têm sido utilizados em vários estudos demonstrando o papel dos múltiplos fatores envolvidos na formação da fronteira entre o Schwann-astrócitos e migração de células de Schwann limitada em ambiente astrocíticos. Compreensão dos mecanismos subjacentes a estes eventos é essencial, uma vez que permitiria o desenvolvimento de estratégias para otimizar e melhorar a integração de enxertos de células de Schwann após o transplante e com isso facilitar a sa...

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		<div>
		<table border="1">
		<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>
		</div>

analysis of schwann-astrocyte interactions using in vitro assays

Células de Schwann são um dos células normalmente usadas em estratégias de reparo após lesões na medula espinhal. Células de Schwann são capazes de apoiar a regeneração axonal e surgimento de secreção 1,2 fatores de crescimento e fornecer factores de crescimento e moléculas de adesão 3 moléculas de matriz extracelular 4. Além disso, eles mielinizar os axônios desmielinizados no local da lesão 5. No entanto após o transplante, as células de Schwann não migram do local de implante e não se misturam com os astrócitos anfitrião 6,7. Isto resulta na formação de uma fronteira nítida entre as células de Schwann e astrócitos, criando um obstáculo para os axônios em crescimento tentando sair da volta do enxerto no tecido do hospedeiro proximal e distalmente. Astrócitos em contato com células de Schwann também sofrem hipertrofia e up-regular as moléculas inibidoras 8-13. Ensaios in vitro têm sido usados ​​para modelar Schwann células-astrócitos interações e têm sido importantes para a compreensão do mecanismo subjacente ao comportamento celular. Estes ensaios in vitro incluem teste de fronteira, onde a co-cultura é feita através de duas células diferentes, com cada tipo de célula que ocupam diferentes territórios, com apenas uma pequena diferença que separa as duas frentes de células. Como as células se dividem e migrar, as duas frentes de celulares se aproximar uns dos outros e, finalmente colidem. Isso permite que o comportamento das duas populações celulares a serem analisados ​​na fronteira. Outra variação da mesma técnica é misturar as duas populações celulares em cultura e ao longo do tempo os dois tipos de células com as células de Schwann segregar aglutinados como ilhas entre astrócitos juntos criando múltiplas Schwann-astrócito limites. Segundo ensaio utilizado no estudo da interação de dois tipos de células é o ensaio de migração celular, onde o movimento pode ser acompanhado na superfície do tipo de célula outras monocamada 14,15. Este ensaio é comumente conhecido como teste de lamela invertido. Células de Schwann são cultivadas em pequenos fragmentos de vidro e eles são invertidos face para baixo sobre a superfície de monocamadas de astrócitos e migração é avaliada a partir da borda da lamínula. Ambos os ensaios têm sido fundamentais para estudar os mecanismos subjacentes envolvidos na exclusão celular e formação de fronteira. Algumas das moléculas identificadas usando estas técnicas incluem N-caderinas, 15 de proteoglicanos sulfato de condroitina (CSPGs) 16,17, FGF / Heparina 18, Ef / Efrinas 19. Este artigo pretende descrever ensaio limite e ensaio de migração de forma gradual e elucidar os possíveis problemas técnicos que possam ocorrer.

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Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Este artigo pretende descrever de forma gradual o comumente usado<em&gt; In vitro</em&gt; Testes usados ​​no estudo de células de Schwann-asrtocyte interações.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

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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Research

JoVE Journal

Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.1K visualizações.  University of Cambridge. Este artigo pretende descrever de forma gradual o comumente usado In vitro Testes usados ​​no estudo de células de Schwann-asrtocyte interações.

Vídeo: 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.

Detecção rápida de Homogêneos Ensaios Biológicos Usando o sistema de modulação Biossensoriais Magnetic

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

Biologia

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.

Análise in vitro de PDZ-dependentes Complexos Macromoleculares CFTR Sinalização

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

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.

Na migração celular in vitro e invasão Ensaios

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&#039; 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.

Análise das reacções de processamento de RNA utilizando células Sistemas gratuitas: 3 &#39;Fim de clivagem de pré-mRNA Substratos<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 Agregação ensaios utilizando hiperfosforilada 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 ...

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.

Análise comparativa da hormona de crescimento humano no soro utilizando SPRI, Nano-SPRI e 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 ...

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.

Medição do metabolismo energético, no tecido da retina explantado, usando análise de fluxo extracelular

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.

Efeito de proteínas fluorescentes na fusão parceiros usando Polyglutamine ensaios de toxicidade em levedura

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.

Análise de microbiota usando PCR de duas etapas e sequenciamento do gene 16S rRNA de próxima geração

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

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 Análise da Função Ligase E3 Ubiquitin

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