Name: dna staining method based on formazan precipitation induced by blue light exposure
Uploaded: 2018-01-28T14:00:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

Este trabalho foi apoiado pela Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt), Chile, projeto 11130263 (para CW), projeto CONICYT, NERC + Programa de Colaboración Internacional PCI-PII20150073 (para CW) e U-inicia a partir do Vicerrectoría de Investigación Universidad de Chile (CW).
Para Tatiana Naranjo-Palma, obrigado pela ajuda na fase inicial deste projecto, Dr. Jorge Babul e Diego Quiroga-Roger para discussão útil, Robert Lesch para revisar o manuscrito e Dirección de ramal de la Facultad de Ciencias Químicas y Farmacêuticas da Universidade do Chile. Graças também ao Marcelo Pérez para edição de vídeo e Neil Stevens para corrigir o texto e Errol Watson para fornecer a voz do narrador.

1. preparação e execução do Gel
<ol><li>Prepare o desnaturação não 12% gel de polyacrylamide gel usando a receita de sódio Dodecil sulfato de sódio-Polyacrylamide Gel eletroforese (SDS-PAGE) encontrados em Sapeka e Russell14 mas usando água em vez de SDS.<ol>
<li>Para preparar a 5 mL de solução de gel, use 1,7 mL de água destilada, 2 mL de acrilamida/bis acrilamida (30/0,8% p/v), 1,3 mL de 1,5 M Tris pH 8,8, 0,05 mL de persulfato de amónio de 10% e 0,002 mL de TEMED.</li>
		<li>Para preparar 2 mL de gel de empilhamento usar 1,5 mL de água, 0,33 mL de acrilamida/bis acrilamida (30/0,8% p/v), 0,25 mL de 1 M Tris pH 6,8, 0,02 mL de persulfato de amónio de 10% e 0,002 mL de TEMED. As dimensões do gel são 7,3 x 8,2 cm x 0,075 cm (l x w x d). Nota: Use um pente bem 15 de 0,75 mm de espessura para a melhor sensibilidade.</li>
	</ol>
</li>
	<li>Adicione as quantidades adequadas de 6x carregando buffer e amostra de DNA para um tubo de microcentrifugadora de 1,5 mL (por exemplo, uso 1 µ l de tampão de carregamento 6 x a 5 µ l de amostra de DNA).</li>
	<li>Coloca o gel na câmara de eletroforese vertical.<ol>
<li>Encha a câmara com 750 mL de 1 tampão de x TAE para executar 2 géis. Para preparar 1 L de 50 x TAE, usar 442 g da base Tris, 57,1 mL de ácido acético glacial e 100 mL de pH de EDTA 0,5 M 8. Adicionar água destilada até um volume final de 1 L.</li>
		<li>Carrega as amostras no gel. Funcione o gel a 100 V por 2 h.</li>
	</ol>
</li>
	<li>Em alternativa, prepare um gel de agarose 1 x TAE usando a receita encontrada na Sapeka14.<ol>
<li>300 ml de 1 x TAE para a câmara de eletroforese horizontal. Funcione o gel por 45 min a 100 V.</li>
	</ol>
</li>
</ol>2. NBT-SG eu coloração (Figura 1).
<ol><li>
Gel do acrilamido coloração.
	<ol>
<li>Preparar as soluções: 0,1% w/v NBT e 1.2 x SG eu. Nota: É possível usar o brometo de etídio, GelRed e SYBR Gold em vez de SG eu, mas eu tive o melhor desempenho de SG.</li>
		<li>Despeje 16,75 mL de 1.2 x SYBR verde eu solução em um recipiente de tamanho apropriado. Nota: Usamos a tampa de uma caixa de ponta da pipeta como um recipiente, e esses volumes são calculados para cobrir o gel usando este recipiente; suas dimensões são 11,6 x 7,6 cm x 2.6 cm (l x w x d).</li>
		<li>Coloque o gel na solução. 3,25 ml de solução de 0,1% w/v NBT dar NBT de 0.2 mM e 1 x (equivalente a 2,0 µM) SG concentrações finais.</li>
		<li>Selar o recipiente com filme plástico e enrole completamente o recipiente com papel de alumínio para bloquear qualquer luz ambiente. Nota: O filme plástico é usado para evitar salpicos e evitar a reação da película de alumínio com a coloração da solução.</li>
		<li>Agitar durante 25 min em um agitador orbital em 60 rpm.</li>
		<li>Remova o alumínio e tampa de plástico. Exponha à luz solar por 90 min máximo. Verificar a cada 5 min até que apareça a banda de interesse.</li>
		<li>Para usar a luz azul, preparar uma protoboard com 3 diodos emissores de luz (LED) e colocar os LEDs 5 mm da superfície do gel (verifique a coloração continuamente, como o tempo necessário dependerá da intensidade da fonte de luz LED).</li>
	</ol>
</li>
	<li>
A coloração do Gel do agarose.
	<ol>
<li>Depois de preparar e executar um agarose 1% gel conforme descrito nas seções 1.4 (as dimensões são 1 x 6,2 cm x 7 cm; l x w x d), siga os mesmos passos como em 2.1 com essas modificações:</li>
		<li>Despeje 41,9 mL de 1.2 x SG eu solução na tampa de uma caixa de ponta da pipeta; suas dimensões são 11,6 x 7,6 cm x 2.6 cm (l x w x d). Adicione 8,1 mL da solução NBT de 0,1%.</li>
		<li>Selar o recipiente com filme plástico e em seguida, enrole completamente o recipiente com papel de alumínio para bloquear qualquer luz ambiente.</li>
		<li>Agitar durante 1 h em um agitador orbital em 60 rpm.</li>
	</ol>
</li>
</ol>3. os dados análise.
<ol><li>
NBT-SG que as curvas de calibração.
	<ol>
<li>Obter imagens de gel de digitalização de 1200 dpi.</li>
		<li>Software aberto para análise de imagem. Endireite as imagens usando um editor de imagem. Execute uma densitometria de imagem.</li>
		<li>Calcule a área sob a curva. Prepare um gráfico do sinal/sinalMax versus concentração de DNA.</li>
	</ol>
</li>
</ol>4. o DNA recuperação.
<ol><li>Preparar um gel de agarose 1% em 1 x TAE usando a receita encontrada na Sapeka14.</li>
	<li>300 ml de 1 x TAE para a câmara de eletroforese horizontal. Carrega 4 µ l de 100 plasmídeo de15 pDJ100 ng / µ l e escada de DNA, seguindo o esquema na Figura 2. Funcione o gel durante 1 h a 100 V.</li>
	<li>Marcar e pesar dois tubos de microcentrifuga de 2 mL.</li>
	<li>Corte o gel no meio para ter duas metades idênticas. Cada um deve conter a escada e o plasmídeo de amostra para comparar as diferentes técnicas de coloração. Uma representação esquemática é na Figura 2.</li>
	<li>Manchar uma metade com NBT-SG eu como na etapa 2.<ol>
<li>Corte a banda do plasmídeo usando um bisturi limpo. Guarde o pedaço de gel em um tubo e pesar.</li>
	</ol>
</li>
	<li>Manchar a outra parte do gel com SG eu com 50 mL de 1 x SG eu solução e agitar lentamente (60 rpm) o gel por 1h na escuridão.<ol>
<li>Coloque o gel em um transiluminador e expor por 2 min. corte a banda do plasmídeo usando um bisturi limpo.</li>
		<li>Guarde o pedaço de gel no outro tubo e pesar. Nota: O protocolo pode ser pausado aqui, mantendo os tubos a-20 ° C.</li>
	</ol>
</li>
	<li>Realize a extração do plasmídeo usando um kit de extração comercial de gel.</li>
</ol>5. plasmídeo normalização.
<ol><li>Em um tubo de microcentrifugadora de 1,5 mL, adicione 10 µ l de plasmídeo extraído e 60 µ l do tipo que da água. Misturar e transferir para uma cubeta de quartzo de 60 µ l ou usar 2 µ l diretamente em um Nanodrop.</li>
	<li>Medir os espectros de absorção entre 230 a 320 nm. Calcule a concentração de plasmídeo da amostra usando a seguinte fórmula: <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> onde D é o factor de diluição aplicado (neste caso é 70/10), Ax é absorção x nm e 50 é o fator de conversão de µ g/mL para dsDNA.</li>
	<li>Dilua as amostras para 9,1 ng / µ l.</li>
	<li>Transformar células competentes de Escherichia coli (e. coli) (por exemplo, XL10 Gold) seguindo a transformação Inoue do procedimento16 usando 5 µ l de amostra diluída.</li>
	<li>Contar o número de colônias sobre os pratos de Petri e observe a diluição. Calcular a colônia formando unidades (CFU) com a seguinte fórmula17: <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> onde D é o factor de diluição, aplicado desde a cultura inicial.</li>
	<li>Execute um teste-t não pareado.</li>
</ol>

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+DNA+using+a+visible+dye,+Nile+blue,+in+electrophoresed+gels.">Detection of DNA using a visible dye, Nile blue, in electrophoresed gels.</a> Anal. Biochem. 280 (2), 322-324 (2000).</li><li>Santill&#225;n-Torres, J. 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R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: a Laboratory Manual. , (2012).</li><li>Hansen, W., Garcia, P. 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Um sensível e romance método de coloração de DNA com base na redução do NBT e o uso do SG, que foi apresentado neste artigo. O passo crítico neste protocolo é o tempo de incubação do gel em NBT-SG eu solução e a concentração de NBT. O tempo de coloração para gel de agarose é maior que para géis de acrilamida por causa da maior espessura de gel de agarose. Esse método não funciona bem na presença de SDS como NBT precipita-se em contacto com ele. Se o gel Obtém um fundo roxo, recomendamos que o gel de...

Com os avanços em bioquímica e biologia molecular, os estudos envolvendo DNA exigiram melhores técnicas para analisar o DNA. O primeiro método para coloração de DNA foi prata coloração, que é muito sensível, mas carece de seletividade e não permite a recuperação de amostra. Mais tarde, o desenvolvimento de coloração fluorescente de DNA permitiu a seletiva quantificação de DNA com a possibilidade de recuperação da amostra. Dentre os corantes fluorescentes primeiros usados para quantificação de DNA foi de brometo de etídio1, que é mutagênico2. No entanto, agora existem corantes fluorescentes melhorados mais seguros e mais sensíveis, tais como GelRed e SYBR Green eu (SG eu)3; Mas todos estes corantes fluorescentes exigem o uso de um transiluminador de (UV) ultravioleta ou fluorímetro.
Existem outras técnicas para coloração visivelmente o DNA, como o azul de metileno4 e violeta cristal5,6,7,8,9, mas todos estes sofrem de sensibilidade reduzida e seletividade. Os sais de tetrazólio são suscetíveis a redução de compostos orgânicos e quando isso acontece eles formam um insolúvel e colorida formazan precipitar10,11. Recentemente, foram mostrados para ligar ao DNA devido seus anéis de tetrazólio positivo12sais de tetrazólio bivalente.
Em uma recente publicação13, foi proposta uma nova técnica visível para manchar e quantificação de DNA em gel de poliacrilamida, usando a redução de um sal de tetrazólio bivalente chamado nitro azul tetrazólio (NBT) e corantes fluorescentes como brometo de etídio, GelRed, SYBR Green I e SYBR Gold. Esta reação funcionou na presença de luz azul ou luz solar e permitiu a recuperação da amostra com qualidade melhorada em comparação com o uso de SG eu com um transiluminador UV. O objetivo deste trabalho é fornecer um protocolo detalhado da técnica de coloração usando tetrazólio sais.

<table><tbody><tr><td>Nitro blue tetrazolium</td><td>Gold Biotechnology</td><td>298-83-9</td><td>reagent used in the staining</td></tr><tr><td>SYBR Green I 10000X</td><td>Thermo-Fisher</td><td>S7563</td><td>reagent used in the staining</td></tr><tr><td>Gel extraction kit</td><td>QIAGEN</td><td>28704</td><td>used to extract plasmid from the gel in integrity assay</td></tr><tr><td>DNA ladder</td><td>Thermo-Fisher</td><td>SM 1331</td><td>used to make calibration curves and as reference to cut band in integrity assay</td></tr><tr><td>Agar Agar</td><td>Merck</td><td></td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>sodium chloride</td><td>Merck</td><td>1064045000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>yeast extract</td><td>Becton, Dickinson and Company</td><td>212750</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>peptone</td><td>Merck</td><td>72161000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>TEMED</td><td>AMRESCO</td><td>761</td><td>used to make polyacrylamide gels</td></tr><tr><td>ammonium persulfate</td><td>Calbiochem</td><td>2310</td><td>used to make polyacrylamide gels</td></tr><tr><td>acrylamide</td><td>invitrogen</td><td>15512-023</td><td>used to make polyacrylamide gels</td></tr><tr><td>bisacrylamide</td><td>SIGMA</td><td>146072</td><td>used to make polyacrylamide gels</td></tr><tr><td>Tris-base</td><td>Merck</td><td>1083821000</td><td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td>used to make TAE buffer</td></tr><tr><td>Acetic acid</td><td>Merck</td><td>1,000,632,500</td><td>used to make TAE buffer</td></tr><tr><td>agarose</td><td>Merck</td><td>16802</td><td>used to make TAE buffer</td></tr><tr><td>spectrophotometer</td><td>Tecan</td><td>infinite 200 pro</td><td>used to quantify DNA plasmid</td></tr><tr><td>water purificatiom unit</td><td>Merck</td><td>Elix 100</td><td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td></tr><tr><td>distilled water</td><td></td><td>-</td><td>used in gels, culture medium, stainings and general solutions</td></tr><tr><td>HCl</td><td>J.T. Baker</td><td>9535-03</td><td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>6X DNA loading dye</td><td>Thermo-Fisher</td><td>R0610</td><td></td></tr><tr><td>5 mm Blue LED</td><td>Jinyuan electronics</td><td>SP-021</td><td>light source used in integrity assay</td></tr><tr><td>protoboard</td><td>KANDH</td><td>KH102</td><td>light source support and circuit&amp;nbsp;</td></tr><tr><td>9 V battery</td><td>Duracell</td><td>-</td><td>used to power the LED&#039;s</td></tr><tr><td>horizontal electrophoresis chamber</td><td>Biorad</td><td>1704486EDU</td><td>used to make and run agarose gel in integrity assay</td></tr><tr><td>vertical electrophoresis kit</td><td>Biorad</td><td>1658002EDU</td><td>used to run polyacrylamide gels in calibration curves</td></tr><tr><td>power supply</td><td>Biorad</td><td>1645050&amp;nbsp;</td><td>used to power the electrophoresis</td></tr></tbody></table>

dna staining method based on formazan precipitation induced by blue light exposure

Métodos de coloração de DNA são muito importantes para a investigação biomédica. Nós projetamos um método simples que permite a visualização de DNA a olho nu, a formação de um precipitado colorido. Ele funciona através da imersão a acrilamida ou gel de agarose DNA em uma solução de 1 x (equivalente a 2,0 µM) SYBR Green eu (SG eu) e 0,20 mM nitro azul tetrazólio que produz um roxo precipitado de formazan quando expostos à luz solar ou especificamente a luz azul. Também, testes de recuperação de DNA foram realizados utilizando um plasmídeo resistente à ampicilina em um gel de agarose corado com nosso método. Obteve-se um maior número de colônias com nosso método do que com a tradicional mancha usando SG eu com iluminação ultravioleta. O método descrito é rápido, específico e não-tóxico para a deteção de DNA, permitindo a visualização de biomoléculas a "olho nu", sem um transiluminador e é barato e apropriado para uso em campo. Por estas razões, o nosso DNA novo método de coloração tem potenciais benefícios para a investigação e a indústria.

Um precipitado de formazan roxo aparece onde se encontra o DNA (verificado por espectrometria de massa13). No experimento, uma escada de DNA foi carregada para fazer uma curva de calibração (Figura 3). O protocolo funciona para gel de agarose e acrilamida, mas tem uma menor intensidade e leva mais tempo em agarose. No entanto, o uso de géis de agarose permite a recuperação da amostra usando um kit disponível comercialmente, e nã...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Este método permite coloração seletiva e quantificação de DNA em géis embebendo o gel em um SYBR Green eu / Nitro azul tetrazólio solução e então exposto à luz solar ou uma fonte de luz azul. Isto produz um precipitado visível e não requer quase nenhum equipamento, tornando-a ideal para uso em campo.

Método baseado na precipitação de Formazan induzida pela exposição à luz azul de coloração de DNA

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

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Biochemistry

9.4K visualizações.  Universidad de Chile. Este método permite coloração seletiva e quantificação de DNA em géis embebendo o gel em um SYBR Green eu / Nitro azul tetrazólio solução e então exposto à luz solar ou uma fonte de luz azul. Isto produz um precipitado visível e não requer quase nenhum equipamento, tornando-a ideal para uso em campo.

Vídeo: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

Eletroforese de DNA usando tiazol laranja em vez de brometo de etídio ou corantes alternativos

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Bioquímica

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescência métodos de microscopia para determinar a viabilidade de bactérias em associação com células de mamíferos

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Imunologia e Infecção

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Técnica High Throughput Fluorometric de Avaliação da macrófago fagocitose e actina Polimerização

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Um sistema integrado para acionar remotamente a transdução do sinal intracelular por Photoactivation Upconversion Nanoparticle-mediada da quinase

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Bioengenharia

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

A Plataforma de Ensaios Anti-biofilme adequado para a Exploração de bibliotecas de compostos naturais

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

Análise de conteúdo de gotas lipídicas em leveduras de fissão e brotamento usando processamento automatizado de imagens

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Inativação de Patógenos via Fotólise de Luz Visível de Riboflavina-5′-Fosfato

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Visualização da resistência bacteriana usando sondas de antibióticos fluorescentes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Viver a microscopia de fluorescência da célula para observar os processos essenciais durante o crescimento de células microbianas

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Biologia

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Rotulagem específica de nucleóides mitocondriais para microscopia estruturada de iluminação com lapso de tempo

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...