1. Etapas de preparação
<ol><li>Prepare o caldo Muller-Hinton (MHB) adicionando 25 g de caldo MH a 1 L de água destilada e misture. Autoclave a 121 °C por 2,5 h. Em seguida, armazene a mídia autoclaved em temperatura ambiente ou na geladeira.</li>
	<li>Subcultura as bactérias em questão (E. coli ESBL) na mídia ágar usando o método de listras de quatro quadrantes e incubar durante a noite a 37 °C.<ol>
<li>Usando um laço estéril, pegue uma colônia e espalhe-a na primeira metade da placa de ágar MacConkey fazendo listras paralelas próximas.</li>
		<li>Usando o laço, espalhe a bactéria no segundo quadrante, listrando-a do primeiro quadrante.</li>
		<li>A partir do segundo quadrante, estenda as raias no terceiro quadrante usando faixas paralelas próximas.</li>
		<li>Espalhe a bactéria no centro do quarto quadrante, espalhando-a do terceiro quadrante.</li>
	</ol>
</li>
</ol>2. Preparação do painel
<ol><li>Coloque quatro placas de 96 poços um ao lado do outro para formar um quadrado. Usando uma fita, grave a bunda deles juntos.</li>
	<li>Repita esta etapa para obter quatro painéis cada um contendo 4 placas e nomeie-os A1, A2, A3 e A4.</li>
	<li>Adicione 50 μL de caldo MH aos poços entre a coluna 2 e a coluna 11 nas 16 placas de 96 poços dos quatro painéis.</li>
	<li>Adicione 200 μL de caldo MH ao poço H12 servindo como o bem de controle negativo nas 16 placas de 96 poços dos quatro painéis.</li>
	<li>Adicione 150 μL de caldo MH aos poços A1 e H1 servindo como poços de controle positivos nas 16 placas de 96 poços dos quatro painéis.</li>
</ol>3. Droga 1, Cefotaxime, diluição em série
<ol><li>A um tubo cônico, adicione 15 mL de dH2O estéril.<ol>
<li>Calcule o volume a ser removido da solução de estoque da droga seguindo a fórmula C1V1 = C2V2. Assim, V1 = (C2 x 15 mL) / C1. NOTA: No nosso caso, a solução de estoque cefotaxime é de 105 μg/mL e C2 é de 256 μg/mL; assim, V1 = 38,4 μL.</li>
	</ol>
</li>
	<li>Remova o volume calculado dos 15 mL de dH2O estéril e adicione a droga.</li>
	<li>Pipeta 50 μL da solução de droga preparada em cada poço na coluna 11 e coluna 12 exceto H12.</li>
	<li>Inicie a diluição seriada removendo 50 μL da coluna 11 e colocando-a nos poços correspondentes na coluna 10 e, em seguida, de 10 até chegar à coluna 2, onde os 50 μL retirados da coluna 2 serão descartados.</li>
	<li>Repita as etapas 3.4 e 3.5 para todas as 16 placas de 96 poços dos quatro painéis.</li>
</ol>4. Droga 2, Amkacina, diluição em série
<ol><li>A um tubo cônico, adicione 10 mL de dH2O estéril.</li>
	<li>Calcule o volume a ser removido da solução de estoque da droga seguindo a fórmula C1V1 = C2V2. Assim, V1 = (C2 x 10 mL) / C1. NOTA: No nosso caso, a solução de estoque amikacin é de 103 μg/mL e C2 é de 64 μg/mL; assim, V1 = 64 μL.</li>
	<li>Remova o volume calculado dos 10 mL de dH2O estéril e adicione a droga.</li>
	<li>Pegue oito placas separadas de 96 poços.</li>
	<li>A cada placa, adicione 100 μL de MHB aos poços entre as linhas G e B.</li>
	<li>Adicione 100 μL da solução de droga 2 previamente preparada aos poços da linha G.</li>
	<li>Diluir serialmente da linha G para a linha B, tomando 100 μL de cada poço e finalmente descartar os 100 μL dos poços da linha B.</li>
	<li>Repita as etapas 4.5, 4.6 e 4.7 para preparar oito placas.</li>
</ol>5. Transferência da droga 2 para os quatro painéis
<ol><li>Pipeta 50 μL de droga 2 dos poços entre as linhas G e B para os poços correspondentes em cada placa nos quatro painéis. Uma placa preparada de 96 poços contém 100 μL de droga 2 que é suficiente para duas placas em um painel.</li>
</ol>6. Droga 3, Levofloxacina, adição
<ol><li>A quatro tubos cônicos diferentes, adicione 14 mL de dH2O estéril.</li>
	<li>Calcule o volume a ser removido da solução de estoque da droga seguindo a fórmula C1V1 = C2V2. Assim, V1 = (C2 x 14 mL) / C1. NOTA: No nosso caso, levofloxacina é preparada em quatro concentrações diferentes de uma solução de estoque de 5 x 103 μg/mL. C1 = 2 μg/mL, V1 = 5,6 μL C2 = 4 μg/mL, V1 = 11,2 μL C3 = 8 μg/mL, V1 = 22,4 μL C4 = 16 μg/mL, V1 = 44,8 μL</li>
	<li>Remova o volume calculado dos 14 mL de dH2O estéril de cada tubo e, em seguida, adicione a droga.</li>
	<li>Após preparar as concentrações necessárias da terceira droga, tome 50 μL e adicione-a aos poços correspondentes entre as linhas B e G e as colunas 2 e 12 na placa correspondente em cada painel onde C1 corresponde às quatro placas P1 nos quatro painéis, C2 corresponde às quatro placas P2 nos quatro painéis , C3 corresponde às quatro placas P3 nos quatro painéis, e C4 corresponde às quatro placas P4 nos quatro painéis.</li>
</ol>7. Droga 4, Trimethoprim-sulfametoxazol, adição
<ol><li>A um tubo cônico, adicione 14 mL de dH2O estéril.</li>
	<li>Calcule o volume a ser removido da solução de estoque da droga seguindo a fórmula C1V1 = C2V2. Assim, V1 = (C2 x 14 mL) / C1. NOTA: No nosso caso, trimethoprim-sulfametoxazol é preparado em quatro concentrações diferentes de uma solução de estoque de 48 x 103 μg/mL. C1 = 512 μg/mL, V1 = 149,33 μL C2 = 1024 μg/mL, V1 = 298,66 μL C3 = 2048 μg/mL, V1 = 597,33 μL C4 = 4096 μg/mL, V1 = 1 194,66 μL</li>
	<li>Remova o volume calculado dos 14 mL de dH2O estéril de cada tubo e, em seguida, adicione a droga.</li>
	<li>Após preparar as concentrações necessárias da quarta droga, tome 50 μL e adicione-a aos poços correspondentes entre as linhas B e G e as colunas 2 e 12 nas quatro placas no painel correspondente onde C1 corresponde ao painel 1, C2 corresponde ao painel 2, C3 corresponde ao painel 3, e C4 corresponde ao painel 4.</li>
</ol>8. Preparação e adição de inóculo bacteriano E. coli ESBL
<ol><li>Usando um laço estéril, transfira uma colônia do isolado bacteriano E. coli ESBL previamente cultivado em uma placa em 2 mL de MHB e vórtice e vórtice.</li>
	<li>Verifique se há turbidez onde deve ser 0,5 McFarland usando um densitômetro.</li>
	<li>Adicione 80 mL de caldo MH estéril a um copo de urina estéril.</li>
	<li>Adicione o inóculo bacteriano do 0,5 McFarland inóculo ao copo de urina seguindo C1V1 = C2V2, onde V1 = (106 x 80 mL) / 108 = 800 μL.</li>
	<li>Pipeta 50 μL da solução inóculo 106 UFC/mL em cada poço, exceto H12, que é o bem de controle de esterilidade.</li>
	<li>Incubar os painéis a 37 °C durante a noite.</li>
	<li>Após a incubação, adicione 50 μL de Iodotetrazolium para registrar o crescimento dos poços.</li>
</ol>9. Protocolo para o modelo FIC (Arquivo Suplementar)
<ol><li>Escreva a maior concentração de droga 1 (Cefotaxime) na célula amarela no painel A.</li>
	<li>Escreva a maior concentração de droga 2 (Amikacina) na célula amarela no painel B.</li>
	<li>Escreva a maior concentração de droga 3 (Levofloxacina) na célula amarela no painel C.</li>
	<li>Escreva a maior concentração de droga 4 (Trimethoprim-sulfametoxazol) no painel D.</li>
	<li>Trace a Linha Vermelha (interface Growth/no Growth) destacando os poços com crescimento em vermelho.</li>
	<li>Escreva o MIC da droga 1 na tabela da droga 1 (ATB1).</li>
	<li>Escreva o MIC da droga 2 na tabela da droga 2 (ATB2).</li>
	<li>Escreva o MIC da droga 3 na tabela da droga 3 (ATB3).</li>
	<li>Escreva o MIC da droga 4 na tabela da droga 4 (ATB4).</li>
	<li>Para calcular FIC para ATB1<ol>
<li>Determine os poços na interface Crescimento/sem Crescimento.</li>
		<li>Na tabela ATB1, clique duas vezes na célula ao lado do FIC1 e arraste a célula amarela à esquerda do painel A para o primeiro bem selecionado.</li>
		<li>Repita o passo 9.10.2 para cada poço selecionado onde bem 1 corresponde ao FIC1, bem 2 corresponde ao FIC2, e assim por diante.</li>
	</ol>
</li>
	<li>Para calcular o FIC para ATB2<ol>
<li>Na tabela ATB2, clique duas vezes na célula ao lado do FIC1 e arraste a célula amarela à esquerda do painel B para o primeiro poço pré-selecionado.</li>
		<li>Repita o passo 9.11.1 para cada poço pré-selecionado.</li>
	</ol>
</li>
	<li>Para calcular o FIC para ATB3<ol>
<li>Na tabela ATB3, clique duas vezes na célula ao lado do FIC1 e arraste a célula amarela à esquerda do painel C para o primeiro poço pré-selecionado.</li>
		<li>Repita o passo 9.12.1 para cada poço pré-selecionado.</li>
	</ol>
</li>
	<li>Para calcular o FIC para ATB4<ol>
<li>Na tabela ATB4, clique duas vezes na célula ao lado do FIC1 e arraste a célula amarela à esquerda do painel D para o primeiro poço pré-selecionado.</li>
		<li>Repita o passo 9.13.1 para cada poço pré-selecionado.</li>
	</ol>
</li>
	<li>Na tabela rotulada ATB1+2+3+4, soma automaticamente o FIC1 de cada ATB. O mesmo ocorrerá para as outras FICs (FIC2, FIC3, e assim por diante).<ol>
<li>Na célula que contém FIC e destacada em amarelo, clique duas vezes nele e selecione os ΣFICs somados da tabela ATB1+2+3+4.</li>
	</ol>
</li>
	<li>Repita estes passos para cada uma das nove folhas representando as nove placas. NOTA: Na folha FIC tudo, a tabela mostrará o FIC somado final com a interpretação do valor obtido.</li>
</ol>10. Determinação mic usando o ensaio de microdiluição para as quatro drogas
<ol><li>Rotule quatro linhas diferentes com a abreviação de cada droga testada. Por exemplo, CTX para Cefotaxime, AMK para Amikacin, LEVO para Levofloxacin e SXT para Trimethoprim-sulfametoxazol.</li>
	<li>Pipeta 200 μL de caldo Muller Hinton estéril em bem número 1 e bem número 12 em cada linha usada. Bem, o número 12 servirá como o controle negativo bem.</li>
	<li>Pipeta 100 μL de caldo Muller Hinton estéril para os poços 2 a 11 em cada linha usada.</li>
	<li>Calcule o volume de cada droga a ser adicionada usando a fórmula C1V1 = C2V2. Assim, V1 = (C2 x 4 x V2) / C1. V2 é o volume final nos poços que é de 200 μL, C1 é a concentração da solução de estoque, e C2 é a concentração inicial que precisamos ter no primeiro poço. NOTA: Aqui, usamos o seguinte: Cefotaxime: C1 = 105 μg/mL, onde o diluímos para 104 μg/mL, C2 = 256 μg/mL; assim, V1 = 20,48 μL Amikacina: C1 = 103 μg/mL, C2 = 64 μg/mL; assim, V1 = 51,2 μL Levofloxacina: C1 = 5 x 103 μg/mL, onde o diluímos para 5 x 102 μg/mL, C2 = 16 μg/mL; assim, V1 = 25,6 μL Trimetópa-Sulfametoxazol: C1 = 48 x 103 μg/mL, C2 = 4096 μg/mL; assim, V1 = 68,26 μL</li>
	<li>Pipeta o volume necessário para cada droga no primeiro poço da linha correspondente depois de remover o mesmo volume do caldo de 200 μL para obter um volume total de 200 μL após a adição da droga.</li>
	<li>Diluir serialmente removendo 100 μL do poço 1 para o poço 2 e assim por diante até chegar bem 10 onde os 100 μL removidos do poço 10 serão descartados. Note que bem 11 serve como o controle positivo bem.</li>
	<li>Pipeta 100 μL de 106 inóculo bacteriano preparado em cada poço em cada linha usada, exceto para o bem número 12 servindo como o controle negativo.</li>
	<li>Incubar a 37 °C durante a noite.</li>
</ol>

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O método De checkerboard quádruplo se assemelha ao tabuleiro de xadrez e ao tabuleiro de damas tridimensional em seu protocolo. No entanto, certas medidas cruciais devem ser tomadas em consideração para evitar erros durante o experimento.
Certifique-se de testar para o MIC de cada droga contra o isolado testado antes de iniciar o protocolo para saber quais são as concentrações necessárias para iniciar as diluições com a droga 1 e a droga 2 que precisam ser diluídas em série nas pla...

Com o aumento da resistência devido ao uso excessivo e uso indevido de antibióticos1,2, a necessidade de desenvolver novos medicamentos e agentes para tratar infecções bacterianas tornou-se crucial. Novas abordagens como o desenvolvimento de novas drogas são muito importantes para superar a crise de resistência. No entanto, a indústria farmacêutica não está interessada em desenvolver novos agentes antimicrobianos. Além disso, se novas drogas forem desenvolvidas, as bactérias continuarão evoluindo e desenvolvendo resistência contra essas novas drogas3,4. Assim, o problema de resistência não será resolvido, tornando a necessidade de outra abordagem uma obrigação que deve ser considerada e estudada para superar a resistência bacteriana.
A combinação de medicamentos é um conceito muito importante para o tratamento de infecções bacterianas principalmente aquelas causadas por patógenos resistentes a multidroga5,6. Diminui o curso do tratamento, diminui a dose dada; assim, a diminuição da toxicidade da determinada droga, ajuda a diminuir a taxa de desenvolvimento de resistência e, de certa forma, sensibiliza a bactéria aos medicamentos fornecidos como descrito no conceito de sensibilidade colateral5,7,8,9.
O desenvolvimento da resistência a uma droga requer uma única mutação; no entanto, o desenvolvimento da resistência a uma combinação de drogas que visam múltiplas vias requer várias mutações independentes que são por essa combinação. Um exemplo para a diminuição da resistência ao usar a terapia combinada é a diminuição da taxa de resistência ao Rifampin no Mycobacterium Tuberculosis10. Outro exemplo é um estudo feito por Gribble et al. que mostrou que a taxa de surgimento de cepas resistentes em pacientes que tomam Piperacillin sozinha para ser maior do que naquelas que tomam uma combinação de carboxipenicilina e aminoglycoside10. Estudos têm demonstrado que o desenvolvimento da resistência aos aminoglicosídeos em bactérias em evolução tornou essas cepas sensíveis a várias outras drogas5. A combinação entre a amoxicilina da droga classe beta-lactam e o ácido clavulanic inibidor de lactamase mostrou sucesso no tratamento de cepas bacterianas resistentes8.
Diminuir o tempo de tratamento é uma boa vantagem resultante de combinações de medicamentos. Por exemplo, uma terapia de penicilina combinada ou ceftriaxona com gentamicina por 2 semanas dará a mesma eficácia dada apenas por penicilina ou ceftriaxona quando dada apenas por 4 semanas11. A combinação de medicamentos permite o uso de doses mais baixas de drogas que não são eficazes quando dadas sozinhas, como os Sub-MICs. O exemplo de sulfonamidas pode ser dado quando o uso de sulfonamidas triplas minimiza, em doses mais baixas, a toxicidade produzida que é a formação de cristais ou cristalizatória ao usar sulfonamidas insolúveis em doses completas12.
Assim, a diminuição da dosagem dada e o tempo de tratamento acabarão por diminuir a toxicidade das drogas no corpo. A ideia de desenvolver métodos para avaliar a interação entre drogas combinadas é muito importante. Em um estudo, os resultados mostraram que a terapia combinada é mais eficaz para o tratamento de espécies resistentes de Acinetobacter e P. aeruginosa8.
Dando drogas em combinação Existem diferentes métodos pelos quais podemos estudar combinações de medicamentos, como o método de tabuleiro de xadrez, o método de curva de morte de tempo e o método de teste E13. O método de checkerboard pode estudar todas as combinações possíveis entre as duas drogas em questão em um experimento em si. Além disso, foi desenvolvido para estudar uma combinação de três fármacos14. Agora, estendemos isso para estudar uma combinação de quatro drogas principalmente para o tratamento de patógenos multidroga resistentes.
O ensaio da curva de morte de tempo é geralmente realizado para testar o efeito bactericida de uma determinada droga. Também foi usado para testar o efeito de combinações de drogas onde várias drogas são combinadas em concentrações específicas. Este protocolo requer a preparação de vários tubos ou copos estéreis onde em cada xícara adicionamos o caldo, a combinação de drogas e a cepa bacteriana necessária. Após a incubação e registro da densidade óptica em vários pontos de tempo, os resultados são comparados com a taxa de crescimento normal da cepa utilizada para ver se a taxa de crescimento aumentou, diminuiu ou não mudou13.
O método de teste eletrônico é geralmente feito para testar a concentração inibitória mínima (MIC) onde uma tira contendo uma concentração gradiente da droga em questão é colocada em uma placa inoculada. Também foi usado para testar a combinação entre duas drogas onde duas tiras são adicionadas à placa de forma perpendicular cruzando seus MICs13.
Segundo a literatura, não há padrão-ouro para definir e estudar sinergia; assim, é difícil avaliar qual dos métodos utilizados para estudar a combinação é melhor e qual produz resultados melhores e mais confiáveis principalmente13. No entanto, o ensaio de morte por tempo é intensivo em trabalho, demorado e caro15,16, enquanto o método de teste eletrônico é desenvolvido para estudar uma combinação entre apenas duas drogas. O Checkerboard pode estudar todas as combinações possíveis entre as duas drogas testadas e é por isso que esta técnica foi escolhida para ser desenvolvida.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330000402</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330-0013-17</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>corning</td>
			<td>430828</td>
			<td>For drug 3 and 4 preparation</td>
		</tr>
		<tr>
			<td>5 mL polysterene round-bottom Tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td>For 0.5 MacFarland bacterial inoculum preparation</td>
		</tr>
		<tr>
			<td>90mm petri dishes</td>
			<td>JRZ Plastilab</td>
			<td></td>
			<td>As bed for the solutions to be added using the multichannel pipette</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>corning</td>
			<td>3596</td>
			<td>For serial diltuion and combining drugs</td>
		</tr>
		<tr>
			<td>Bactrim 200, 40 mg (Trimethoprim-sulamethoxazole</td>
			<td>By CRNEXI SAS Fontenay-sous-Bois, France</td>
			<td>10177403</td>
			<td>Drug 4</td>
		</tr>
		<tr>
			<td>Ceforane, 1 g (Cefotaxime)</td>
			<td>PHARCO Pharmaceuticals</td>
			<td>24750/2006</td>
			<td>Drug 1</td>
		</tr>
		<tr>
			<td>Densitometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. Coli ESBL strain</td>
			<td>Retreived as a medical strain from the Saint-George Hospital Lebanon</td>
			<td></td>
			<td>Bacterial strain</td>
		</tr>
		<tr>
			<td>Mac Conkey + crystal violet agar</td>
			<td>BIO-RAD</td>
			<td>64169508</td>
			<td>For making agar plates used for subculturing</td>
		</tr>
		<tr>
			<td>Miacin 500 mg/2 mL (Amikacin)</td>
			<td>HIKMA Pharmaceuticals</td>
			<td>2BXMIA56N-AEF</td>
			<td>Drug 2</td>
		</tr>
		<tr>
			<td>Muller-Hinton Broth</td>
			<td>BIO-RAD</td>
			<td>69444</td>
			<td>For making bacterial media</td>
		</tr>
		<tr>
			<td>Multichannel Pipette</td>
			<td>Thermo Scientific</td>
			<td>GJ54761</td>
			<td>For serial dilution and addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Paper Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel pipettes</td>
			<td>Thermo Scientific</td>
			<td>OH19855 HH40868</td>
			<td>For the addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Tavanic, 500 mg (Levofloxacin)</td>
			<td>sanofi aventis</td>
			<td>221937/2009</td>
			<td>Drug 3</td>
		</tr>
	</tbody>
</table>

quadruple-checkerboard: a modification of the three-dimensional checkerboard for studying drug combinations

O conceito de terapia de combinação de drogas está se tornando muito importante principalmente com o aumento drástico da resistência às drogas. O tabuleiro de xadrez Quádruplo, também chamado de Q-checkerboard, visa maximizar o número de combinações possíveis que podem ser obtidas entre quatro drogas em um experimento para minimizar o tempo e o trabalho necessários para alcançar os mesmos resultados com outros protocolos. Este protocolo baseia-se na simples técnica de micro diluição onde as drogas são diluídas e combinadas em várias placas de 96 poços.
No primeiro conjunto de placas de 96 poços, o caldo Muller-Hinton é adicionado seguido pela primeira droga necessária (por exemplo, Cefotaxime aqui) para diluí-lo em série. Após o primeiro passo, outro conjunto de placas de 96 poços é usado para diluir a segunda droga (por exemplo, Amikaci), que será transferida removendo um volume específico de droga 2 e colocada nos poços correspondentes no primeiro conjunto de placas de 96 poços que contém uma droga. O terceiro passo é feito adicionando as concentrações necessárias da terceira droga (por exemplo, Levofloxacin), às placas apropriadas no conjunto inicial contendo combinação de droga 1 e 2. O quarto passo é feito adicionando as concentrações necessárias da quarta droga (por exemplo, Trimethoprim-sulfamethoxazol) nas placas apropriadas no primeiro conjunto. Em seguida, o inóculo bacteriano E. coli ESBL será preparado e adicionado.
Este método é importante para avaliar todas as combinações possíveis e tem uma gama mais ampla de possibilidades a serem testadas além para testes in vivo. Apesar de ser uma técnica cansativa que requer muito foco, os resultados são notáveis e economizam tempo onde muitas combinações podem ser testadas em um único experimento.

A Figura 2A representa os resultados obtidos pela combinação de Cefotaxime e Amikacin com concentrações específicas de Levofloxacina e Trimetoprim-sulfametoxazol. Podemos ver na parte esquerda da figura as quatro placas que são esquemáticamente apresentadas com as concentrações das drogas na parte direita da figura. As setas representam os poços na interface Crescimento/sem Crescimento. Os poços coloridos são os poços que contêm crescimento. Notamos que a quarta placa não cont...

Watch this Scientific Journal Video about Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations at JoVE.com

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

Este protocolo descreve como estudar todas as combinações possíveis que podem ser obtidas entre quatro drogas em um único experimento. Este método baseia-se no ensaio padrão de micro diluição de placas de 96 poços e no cálculo de concentrações inibitórias fracionárias (FICs) para avaliar os resultados.

Quadruplicar-Checkerboard: Uma modificação do tabuleiro de xadrez tridimensional para estudar combinações de medicamentos

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

quadruple-checkerboard-modification-three-dimensional-checkerboard

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4.4K visualizações.  University of Balamand. Este protocolo descreve como estudar todas as combinações possíveis que podem ser obtidas entre quatro drogas em um único experimento. Este método baseia-se no ensaio padrão de micro diluição de placas de 96 poços e no cálculo de concentrações inibitórias fracionárias (FICs) para avaliar os resultados.

Vídeo: Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Chip baseado em Cultura de Células tridimensional em perfundidos Micro-biorreatores

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biologia

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabricação de Chip porte Andaimes para tridimensional de cultivo celular

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Oi-C: um método para estudar a arquitetura tridimensional de Genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

Uma ferramenta analítica que quantifica Alterações morfologia celular de imagens tridimensionais de fluorescência

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Usando tomografia de tórax para visualizar a estrutura tridimensional e Função da Usina Vasculatura

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

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 ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Reconstrução tridimensional da arquitetura Vascular do ovário passiva Mouse clareza-limpo

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Um método de compensação por imagem tridimensional do tecido adiposo tecido

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Modificação genética de cianobactérias por conjugação usando o kit de ferramentas de clonagem modular CyanoGate

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Resolução unicelular imagens tridimensionais de organoides intactos

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...