Os autores gostariam de agradecer: Dr. Frank Jiang, Dr. David Lin, Dr. Bernard Yurke e Dr. Uday Chippada por suas contribuições sobre o desenvolvimento da tecnologia do gel de DNA; Dr. Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter pelos seus comentários e edições deste manuscrito; fontes de financiamento, incluindo a Comissão Jersey New on Spinal Cord Research (Grant # 07A-019-SCR1, NAL) e New Jersey Institute Neuroscience (MLP); e editores de Engenharia de Tecidos, Parte A permissão para reimprimir Figuras 2 e 4 e Biomateriais permissão para reimprimir figura 3.

<ol>
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Os autores não têm nada a revelar.

A capacidade dos geles de ADN para amolecer ou endurecer antes e após a adesão das células torna-os um modelo ideal para estudar o papel da rigidez do tecido dinâmico em função da célula. Todos os três modelos têm sido utilizados em estudos biológicos e mecânicos. No entanto, todos os três modelos têm elasticidade semelhantes em várias percentagens de reticulação, que indica o comprimento de reticulação não influenciar a elasticidade de gel do DNA (Tabela 2). Em contraste, a concentra...

Substratos estáticos e dinâmicos são duas categorias de biomateriais que foram desenvolvidas para estudar os efeitos de elasticidade do tecido, ou em função da rigidez das células. Substratos estáticos são incapazes de alterar as suas propriedades físicas depois de serem fabricadas e / ou uma vez as células são banhados. Poliacrilamida (PA) foram os primeiros géis bidimensionais substratos, estáticas que foram sintetizadas para investigações mechanobiology 5,17. Geles PA são fáceis de preparar, barato, versátil e pode ser fabricado com uma ampla gama de módulos elásticos. Embora essas vantagens técnicas fazem PA géis um substrato comumente aplicado, substratos estáticas não são indicativos da natureza dinâmica da matriz extracelular (MEC) e ao ambiente circundante celular in vivo. Por exemplo, a rigidez ECM sofre alterações em resultado de ferimento, de desenvolvimento, ou doença. Substratos dinâmicos são, portanto, preferidas como substratos modelo mimetizando a tecidos em estudos mechanobiology <sup&gt; 22,24,25. Numerosos sintético, Bidimensional biomateriais, tridimensionais, estáticos e dinâmicos naturais têm sido desenvolvidos para imitar rigidez tecidual 1,3,6,16,23,26. Alguns substratos dinâmicas exigem calor, UV, corrente elétrica, os íons, e mudanças de pH de alterar as suas propriedades mecânicas 2,4,7,8,12,15,16, mas esses estímulos podem restringir bio-aplicação do hidrogel. Hidrogeles de poliacrilamida reticulada-DNA (DNA) são géis elásticos substratos bidimensionais dinâmicas. Ligações cruzadas de DNA permitem a modulação temporais, espaciais, e reversível da rigidez gel DNA pela adição de DNA de fita simples (ssDNA) para mídia ou buffer 9-11,13,14,18,21. Ao contrário dos géis dinâmicas acima mencionados, onde são aplicados estímulos para modulação de elasticidade, os géis de DNA contam com a difusão de ADNcs aplicada para a alteração da elasticidade. Por conseguinte, a superfície superior de gel, em que as células são cultivadas, é a primeira área modulada, porque a taxa de omodulação f elasticidade está dependente da espessura do gel. Geles de DNA são semelhantes aos seus homólogos gel PA na medida em que têm uma espinha dorsal de poliacrilamida, no entanto, as ligações cruzadas de bis-acrilamida substituída com ligações cruzadas são compostos por ADN (Figura 1). Dois ssDNAs (SA1 e SA2) hibridar com uma cadeia reticulador (L2) para compensar as ligações cruzadas de DNA do gel. SA1 e SA2 têm seqüências distintas que ambos contêm uma modificação Acrydite no final 5 &#39;para a incorporação efetiva na rede PA. Para a preparação do gel, SA1 e SA2 são polimerizados, individualmente, em uma espinha dorsal PA e, subsequentemente, o SA1 polimerizada e SA2 são misturados em conjunto. L2, o agente de reticulação, é adicionado à mistura de SA1 e SA2. A sequência de bases é complementar a L2 ambas as sequências SA1 e SA2 e L2 hibrida com SA1 mais SA2 para formar as ligações cruzadas de DNA. Inicial, a elasticidade de gel de ADN é determinada por ambas as concentrações de L2 e de reticulação (Tabelas 1 </strong&gt; e 2). Geles de ADN que contenham quantidades estequiométricas iguais de L2, AE1, AE2 e os géis são mais duras, porque SA1 e SA2 são 100% reticulado por L2 (designado como 100% de gel). Baixas concentrações do resultado L2 em uma menor percentagem de reticulação DNA e, portanto, géis de DNA mais suave. Géis tão baixas como 50% reticulado (designado como 50% de gel) 9-11 foram construídas. <img alt="Figura 1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> A Figura 1 de reticulação de gel do DNA e uncrosslinking esquemática 9-11,13,14,18,21 Etapa 1:. SA1 (vermelho) e SA2 (azul) são polimerizados, individualmente, em uma espinha dorsal de poliacrilamida (preto). Após a polimerização, as soluções polimerizados SA1 e SA2 são misturados em conjunto. Passo 2: L2 (verde) é adicionado e hibrida com SA1 mais SA2 para formar as ligações cruzadas do gel. Passo 3: R2 hibrida com tele toehold de L2. Passo 4: hibridização toehold de R2 impulsiona a descompactação de L2 de SA1 e SA2. Ao contrário dos géis PA, géis de DNA pode endurecer e amaciar após a síntese. Por essa razão, as células cultivadas em géis de ADN pode ser submetido a alterações de rigidez dinâmica. Para endurecer géis de células aderentes, L2 pode ser adicionado ao meio de cultura de baixo géis percentuais, para aumentar a percentagem de ligações cruzadas. Para suavizar a géis de células aderentes, L2 podem ser removidas para reduzir a percentagem de ligações cruzadas 10,13,21. L2 tem uma sequência de ponto de apoio adicional na extremidade 3 &#39;para permitir a L2 uncrosslink de SA1 e SA2 (Tabela 1). Remoção de L2 é realizado através de hibridação de um fio chamado inversão R2. R2 é complementar ao comprimento total de L2 e hibrida com o primeiro ponto de apoio L2. Toehold hibridação impulsiona a descompactação de L2 de SA1 e SA2, que elimina a ligação transversal e reduz a rigidez do gel. Neste relatório evídeo, passo-a-passo as instruções são fornecidas para a preparação de endurecimento e amolecimento géis de DNA. Embora 100% e de 80% as preparações de gel são descritas, este protocolo pode ser adaptada para criar ADN de géis reticulados outras percentagens iniciais e finais. Em geral, 100% e 80% de gel são preparadas, imobilizada em lamelas de vidro, funcionalizada, e semeadas com células. L2 é adicionado à media de 80% de gel e R2 é adicionado aos meios de comunicação de 100% de gel, 48 horas após o plaqueamento. A adição de meios de L2 para endurece 80% de gel de ligação cruzada a 100%, ao passo que a adição de R2 a mídia amolece a 100% de gel de 80% de ligações cruzadas. Geles endurecidos são designados como 80 → 100% de gel e geles suavizados são designados como 100 → 80% de gel no texto. Para o controlo ou géis estáticos, ADNcs ou consistindo em Ts Como é entregue a outro conjunto de 100% e 80% de gel. Depois de um mínimo de dois dias seguintes modulação elasticidade, as células podem ser processados ​​e analisados. <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page = &quot;always&quot;&gt; <tbody align="center"><tr><td rowspan="2"></td><td rowspan="2"> Reticulação DNA </td><td rowspan="2"> # De bases </td><td> Sequence (ponto de apoio) </td><td rowspan="2"> Modificação </td><td rowspan="2"> A temperatura de fusão (T m, ° C) </td><td rowspan="2"> Comentários </td></tr><tr><td> 5 &#39;→ 3&#39; </td></tr><tr><td rowspan="4"> Projeto 1 </td><td> SA1 </td><td> 10 </td><td> GCA CCT TTG C </td><td> 5 &#39;Acrydite </td><td> 34,9 </td><td></td></tr><tr><td> SA2 </td><td> 10 </td><td> GTC AGA ATG A </td><td> 5 &#39;Acrydite </td><td> 23,6 </td><td></td></tr><tr><td> L2 </td><td> 30 </td><td> TCA TTC TGA C GC AAA GGT GC G CTA CAC TTG </td><td></td><td> 56 </td><td>Uma sequência de 10 pb toehold está incluído. </td></tr><tr><td> R2 </td><td> 30 </td><td> CAA GTG TAG CGC ACC TTT GCG TCA GAA TGA </td><td></td><td></td><td> R2 é complementar a L2 </td></tr><tr><td rowspan="4"> Projeto 2 </td><td> SA1 </td><td> 14 </td><td> CGT GGC ATA GGA CT </td><td> 5 &#39;Acrydite </td><td> 46.9 </td><td></td></tr><tr><td> SA2 </td><td> 14 </td><td> GTT TCC CAA TCA GA </td><td> 5 &#39;Acrydite </td><td> 40,2 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCT GAT TGG GAA AC A GTC CTA TGC CAC G GT TAC CTT CAT C </td><td></td><td> 65.9 </td><td> A seqüência toehold 12 bp está incluído. </td></tr><tr><td> R2 </td><td> 40 </td><td> GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAG A </td><td></td><td> 65.9 </td><td> R2 é complementar a L2 </td></tr><tr><td rowspan="3"> Design 3 </td><td> SA1 </td><td> 20 </td><td> ACG GAG GTG TAT GCA ATG TC </td><td> 5 &#39;Acrydite </td><td> 55 </td><td></td></tr><tr><td> SA2 </td><td> 20 </td><td> CAT GCT TAG GGA CGA CTG GA </td><td> 5 &#39;Acrydite </td><td> 56.6 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCC AGT CGT CCC TAA GCA TG G ACA TTG CAT ACA CCT CCG T </td><td></td><td> 68.8 </td><td> Ponto de apoio não está incluído. </td></tr><tr><td rowspan="2"> Controle </td><td rowspan="2"> Controle </td><td rowspan="2"> 20-40 </td><td> AAA AAA (etc.) ou </td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td> TTT TTT (etc.) </td></tr></tbody></table> Tabela 1. seqüências de bases para ssDNA 9-11,13,14,18,21. Celular e estudos mecânicos têm utilizado vários dmodelos de reticulação iferentes para gerar géis de ADN com uma variedade de propriedades mecânicas estáticas e dinâmicas. Os parâmetros modulados em design de reticulação são sequência de bases de comprimento e sequência ou comprimento de reticulação. Fontes em negrito e itálico ilustrar o pareamento de bases entre SA1 e L2 e entre SA2 e L2, respectivamente. <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody align="center"><tr><td rowspan="2"></td><td colspan="8"> Projeto </td></tr><tr><td colspan="3"> 1 </td><td colspan="3"> 2 </td><td colspan="2"> 3 </td></tr><tr><td> Concentração de acrilamida (%) </td><td colspan="3"> 10 </td><td colspan="3"> 10 </td><td> 10 </td><td> 4 </td></tr><tr><td> SA1 mais SA2 hibridado com L2 (% reticulado) </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 100 </td><td> 100 </td></tr><tr><td> &lt;strong&gt; Elasticidade (kPa, média ± SEM) </td><td> 6,6 ± 0,6 </td><td> 17,1 ± 0,8 </td><td> 29,8 ± 2,5 </td><td> 5,85 ± 0,62 </td><td> 12,67 ± 1,33 </td><td> 22,88 ± 2,77 </td><td> 25,2 ± 0,5 </td><td> 10,4 ± 0,6 </td></tr></tbody></table> Tabela 2 o módulo de Young (E) de geles de ADN. 9-11,13,14,18,21 concentração de acrilamida, percentagem de ligações cruzadas, e comprimento de ligação transversal pode ser modulada em geles de ADN. Designs 1, 2 e 3 têm 20, 28 e 40 pb comprimentos de ligação transversal, respectivamente. 100% de gel para todos os projetos têm módulos semelhantes indicando comprimento reticulação não afeta a elasticidade gel. No entanto, as variações na concentração de acrilamida alterar a elasticidade em gel de ADN.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

Mechanobiology é uma área científica emergente que aborda o papel crítico de pistas físicas em dirigir a morfologia ea função das células. Por exemplo, o efeito da elasticidade do tecido em função celular é uma importante área de pesquisa por causa da rigidez do tecido mechanobiology modula com a doença, o desenvolvimento, e a lesão. Materiais imitando tecido-estática, ou materiais que não podem alterar a rigidez vez células são banhados, são predominantemente utilizados para investigar os efeitos da rigidez do tecido sobre as funções celulares. Embora as informações recolhidas a partir de estudos estáticos é valiosa, esses estudos não são indicativos da natureza dinâmica do microambiente celular in vivo. Para melhor enfrentar os efeitos da rigidez dinâmica em função das células, foi desenvolvido um sistema de poliacrilamida hidrogel reticulado-DNA (géis de DNA). Ao contrário de outros substratos dinâmicos, géis de DNA tem a capacidade de aumentar ou diminuir a rigidez após a fabricação, sem estímulos. Geles de ADN consistem de ADN crosslAs tintas que são polimerizados em uma espinha dorsal de poliacrilamida. Adição e remoção de ligações cruzadas através da entrega de DNA de fita simples permite temporais, espaciais e controle reversível da elasticidade gel. Temos demonstrado em relatórios anteriores que a modulação de elasticidade dinâmico gel DNA influencia o comportamento de fibroblastos e neurônio. Neste relatório e vídeo, nós fornecemos um esquema que descreve os mecanismos de reticulação do gel de DNA e instruções passo-a-passo sobre os géis de preparação de DNA.

Antes de nossos estudos, interações célula-ECM foram observados em biomateriais compatíveis estáticas ou dinâmicas em substratos irreversíveis e unidirecionais. Estes substratos não refletem com precisão a natureza dinâmica do microambiente celular. Nosso trabalho muda os paradigmas técnicos existentes, fornecendo um modelo mais fisiológico para estudar as interações célula-ECM no amolecimento e endurecimento biomateriais dinâmicos. Em estudos anteriores, foram analisados ​​os efeitos de substratos d...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

O nosso laboratório desenvolveu hidrogéis de poliacrilamida reticulada-ADN, um sistema dinâmico de hidrogel, para compreender melhor os efeitos da modulação da rigidez do tecido sobre a função das células. Aqui, nós fornecemos esquemas, descrições e protocolos para preparar estes hidrogéis.

Preparação de ADN de poliacrilamida reticulada-hidrogéis

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.4K visualizações.  JFK Medical Center.  O nosso laboratório desenvolveu hidrogéis de poliacrilamida reticulada-ADN, um sistema dinâmico de hidrogel, para compreender melhor os efeitos da modulação da rigidez do tecido sobre a função das células. Aqui, nós fornecemos esquemas, descrições e protocolos para preparar estes hidrogéis. 

Vídeo: Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Rápido e sensível Colloidal Coomassie G-250 coloração para proteínas em gel de poliacrilamida

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Biologia

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Desnaturação Uréia Eletroforese em Gel de Poliacrilamida (PAGE Urea)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

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.

O encapsulamento celular em Hidrogéis 3D para Engenharia de Tecidos

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

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Eletroforese em Gel de Poliacrilamida azul Nativo (BN-PAGE) para Análise de Complexos Multiproteicos de Lisados ​​Celular

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

A estimulação mecânica de condrócitos-agarose Hidrogéis

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging no hidrogéis tridimensionais

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

Hidrogéis alginatos desordeiros descabidos proteolíticos e microbiorreatores hidrofóbicos para encapsulamento de oócito suíno

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Detecção de glicosaminoglicanos por Electrophoresis gel poliacrilamida e coloração de prata

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Um ensaio de quimiotaxis melhorado para a identificação rápida de quimioattractants rizobacterianos em Exudates radiculares

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Incorporando reações de CFPS em uma variedade de matrizes de hidrogel

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Autor em destaque: uma nova abordagem para incorporar reações de síntese de proteínas livres de células em hidrogéis 

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Métodos para Incorporação de Reações de Síntese Proteica Livre de Células em Hidrogéis em Macroescala

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...