Este estudo foi apoiado pelo United States Department of Agriculture, Agricultural Research Service. nomes comerciais ou produtos comerciais nesta publicação são mencionados somente para a finalidade de fornecer informações específicas e não implica qualquer recomendação ou conselho pelo Departamento de Agricultura dos Estados Unidos. O USDA é um fornecedor de oportunidades iguais e empregador.

1. O Fringe periférica de origem bacteriana Colony <ol><li> Crescer X. fastidiosa (Xf) Temecula 22 de tipo selvagem, mutante de deleção pilG Xf ΔpliG (usando estratégia de deleção anteriormente descritos 23), e a sua complementar XfΔpliG -C (usando previamente descrito estratégia de complementação genética baseada no cromossoma 24) na PD2 de agar com meio placas 25 a 28 ° C durante 5-7 dias. </li><li> Celofane autoclave (1 x 1 cm2) em água a 121 ° C (249 ° F) durante 15 min. Pegue um pedaço de celofane, drenar a água tocando em um canto do celofane em uma placa de Petri vazia, com cuidado colocar o celofane mais de 15% da superfície de agar e ar seco. </li><li> Pegar indivíduo X. colónias fastidiosa com estéril palitos arredondadas e células no local assepticamente em uma folha esterilizada de celofane sobreposto ao 15% da superfície do agar nas placas de ágar. Incubar a placas a 28 ° C durante 2-3 dias. </li><li> Examinar a morfologia da borda das colónias utilizando um microscópio de dissecação com uma lente objectiva 2X e uma lente ocular 10X. Fotografar a orla periférica em torno de colónias. </li></ol> 2. Microscopia e microfluídicos Fluxo Chambers <ol><li> Fabricar dispositivos microfluídicos utilizando procedimentos de foto-litográfica semelhantes aos descritos anteriormente 5,18. Projetar quatro canais paralelos com software de desenho assistido por computador sobre silício mestre usando métodos de litografia padrão 26. </li><li> Criar as câmaras microfluídicos de mestre de wafer de silício com polidimetilsiloxano (PDMS). Pour PDMS não polimerizados sobre a bolacha de silício mestre e curá-la a 60 ° C durante 1 h. Descole a réplica de PDMS do mestre wafer e aparar a réplica PDMS com uma lâmina em um 22 mm x 40 mm como o mesmo tamanho de uma lamela de vidro. </li><li> Expor a réplica de PDMS, uma lamela de vidro (22 x 40 mm2), e um microscslides ope (51 x 76 mm2) ao plasma de ar a 30 W por 2 min 27. Sanduíche do corpo PDMS entre a lamela de vidro eo vidro de microscópio para construir a câmara uidic fl micro. </li><li> Perfurar um furo (5,5 mm de diâmetro) através do PDMS em cada extremidade do canal modelado. Corte o tubo de borracha de silicone em 12-20 cm de comprimento. Inserir uma extremidade da tubagem de borracha de silicone (5,1 mm de diâmetro externo, 2,1 mm de diâmetro interno, parede de 0,8 mm) em cada extremidade dos canais da réplica de PDMS de abertura, e selá-lo com PDMS não polimerizados a 60 ° C durante 1 h. </li><li> Ligue a outra extremidade do tubo até o fim farpado de conectores luer de plástico. Enrole as câmaras de microfluidos montadas com a folha de alumínio e autoclave los durante 20 min. </li><li> Recolha células de bactérias de X. fastidiosa tipo selvagem, mutante Xf ΔpliG, e complementar XfΔpliG -C através de raspagem, usando inoculação descartáveis ​​laços de placas de meio PD2. Ajustar a densidade celularpara uma densidade óptica de 0,05 a 600 nm em PD2 caldo como descrito anteriormente 23. Recolhe-se o solução da célula bacteriana numa seringa de 1 ml estanque ao gás. </li><li> Montar o dispositivo microfluídico em um palco microscópio invertido. Ligue um tubo de entrada para a seringa estanque ao gás 5 ml contendo PD2 caldo. Montar a 5 ml estanque ao gás seringa com as bombas de seringa. </li><li> Ligue o tubo de saída a um reservatório de resíduos. Manter uma taxa de fluxo de meio de 0,2 mL min -1 durante 30 minutos para estabilizar o sistema. </li><li> Conectar tubos laterais de entrada a uma seringa de 1 ml estanque ao gás que contém a solução da célula bacteriana. Lavar a solução de células bacterianas através do tubo de borracha até que o canal seja atingido. Manter uma taxa de fluxo de meio de 0,2 mL min -1 durante mais 30 minutos, a fim de lavar as células não ligadas da câmara antes da captura da imagem. </li><li> Montar o obturador microscópio sob a parte ajustada ao campo do microscópio para controlar a luz. Ligue o obturador para a co obturadorntrol sistema e ligar o sistema de controlo do obturador para o computador. </li><li> Montar uma câmera digital à porta de vídeo do microscópio e conecte-o ao computador. Executar o software de gravação de lapso de tempo, selecione a função &quot;obturador&quot; do menu, e reconhecer automaticamente o obturador instalado como padrão no software para estabelecer conexões com o obturador com o software. </li><li> Seleccione a função &quot;camera digital&quot; a partir do menu do software de gravação de lapso de tempo para reconhecer automaticamente a câmera digital como o dispositivo de captura padrão no software e estabelecer a comunicação com a câmera digital com o software. </li><li> Localizar as células bacterianas em um dos canais usando 20X de contraste de fase óptica e, em seguida mudar para a lente da objectiva de 40X, antes da captura da imagem. </li><li> Executar o software de gravação de lapso de tempo, selecione a função &quot;aquisição de imagem&quot; usando parâmetros padrão a partir do menu para adquirir as imagens do microscópio.Em seguida, abra a função &quot;Adquirir time-lapse&quot; e defina o intervalo de tempo para 30 seg 5,18,28, com duração de 6-24 horas, dependendo experiência necessária para observar se contraindo motilidade dos X. fastidiosa 5,18,28. Clique em &quot;OK&quot; para iniciar a gravação de lapso de tempo. Clique em &quot;função de pilha&quot; a partir do menu, selecione &quot;salvar como&quot; para empilhar as imagens na pasta de destino no computador depois de terminar a gravação. </li><li> Para múltiplos canais, capturar imagens de lapso de tempo a partir do primeiro canal a cada 30 seg durante 6 horas. Mover a lente objectiva do microscópio para o próximo canal para localizar as células alvo. Repita a função de lapso de tempo, como descrito acima para capturar imagens em cada um dos quatro canais sequencialmente se experimento está configurado para utilizar quatro canais. Continue a captura de imagens de lapso de tempo para até três dias consecutivos. Todas as experiências foram realizadas à temperatura ambiente (23 ± 2 ° C). </li><li> Compilar as imagens de lapso de tempo em umarquivo de vídeo usando o software de imagem de gravação de lapso de tempo. Executar o software de gravação de lapso de tempo, clique em &quot;função Stack&quot; no menu e selecione &quot;função de pilha aberta&quot; para abrir os arquivos empilhados a partir do computador. </li><li> Inicie a função de &quot;fazer cinema&quot; do módulo de pilha, a seleção de todas as imagens e escolher o formato de saída &quot;AVI&quot;. Clique em &quot;Salvar como&quot; para armazenar o arquivo de vídeo na pasta de destino no computador. </li><li> Seleccione os filmes compilados a partir da pasta de destino no computador e reproduzi-los. Em seguida, observe a motilidade das células individuais através da visualização resultante da atividade espasmos motilidade de células de bactérias nos arquivos de vídeo gerados. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Neste estudo, caracteriza o comportamento de movimento de X. fastidiosa PilG mutante Xf ΔpilG e suas complementares estirpes XfΔpilG- C em recém-projetado de múltipla nano-canal paralelo micro câmaras fl uidic. As câmaras uidic recém-projetado micro fl pode ter até quatro câmaras paralelas com 100 um nano-canal de largura em comparação com modelos anteriores, com apenas uma única de 50 m de largura do canal 18. A melhoria mais ampla nano-canal facilita ...

Xylella fastidiosa é uma bactéria patogénica gram-negativas não flagelados, que faz com que um certo número de doenças de culturas economicamente importantes, incluindo a doença de Pierce em videira (Vitis vinifera L.) 1,2, 3. Esta bactéria é limitada ao xilema de condução de água embarcações. Infecção da videira faz com que o bloqueio dos vasos do xilema e resulta em estresse hídrico e deficiências nutricionais 3. Colonização bem sucedida depende da capacidade da bactéria para se mover a partir do local inicial da infecção para o resto da planta 3. Espasmos motilidade é um meio de movimento bacteriana independente de flagelar através da extensão, apego e retração do pili tipo IV polar 4 que tem sido caracterizado X. fastidiosa 5,6,7. A motilidade espasmos tem sido observado por pinças laser e microscopia de força atômica (AFM) 8,9,10. Utilizando estas técnicas, tmotilidades witching gerado pelo pilus de tipo IV de N. gonorrhoeae e P. aeruginosa foram caracterizados por fl pili rotulagem uorescently e capturando os seus movimentos microscopicamente. Embora ambos os métodos tenham detalhado a força adesiva de bactérias individuais, os procedimentos são complicados e demorados 9,10. As câmaras uidic fl micro foram usados ​​para observar a migração de longa distância de células individuais, bem como pequenos agregados de células bacterianas 5,6. Estas câmaras foram concebidas como um-nano-canal microfabricated em uma placa integrada com um lapso de tempo 11,12,13,14 sistema de gravação de imagem. Micro fl dispositivos câmara uidic oferecem várias vantagens para estudar o comportamento movimento e célula-célula interações de bactérias: (i) fornece uma plataforma integrada com múltiplas capacidades de canal; (Ii) que pode examinar os movimentos e agregações de células individuais nas características de nano-escala de bactérias; (Iii) que permite m diretaicroscopic gravação de imagem de células bacterianas e análises de lapso de tempo, (iv) ele fornece observações espaciais e temporais de longo prazo de populações individuais e / ou de bactérias em um micro-ambiente; (V) a taxa de fluxo de meio de cultura em um canal pode ser controlada com precisão e (VI), apenas um muito pequeno volume (1 ml) de meio de cultura é necessário para cada experiência. Recentemente, o micro fl uidic fluxo do sistema foi utilizado para investigar o comportamento de células bacterianas sob diferentes microambientes 14,15,16. A adesividade e o anexo de superfície do E. coli 15, X. fastidiosa 16 e Acidovorax citrulli 14 em superfícies de vidro foram avaliados através de câmaras uidic micro fl. A formação de agregação e biofilme mediada por tipo pili IV do Acidovorax citrulli foram analisados ​​14. Além disso, o movimento de A. citrulli observado sob fl uxo cs condições demonstraram que o pili de tipo IV podem desempenhar papéis importantes na colonização e a propagação de A. citrulli nos vasos do xilema sob seiva fluxo condições. Os motilidades espasmos de Pseudomonas aeruginosa e X. fastidiosa células foram observadas com sucesso contra uma corrente de fluido numa câmara de fluxo microfabricado 5,6,17. Pilus de tipo IV deficiente PilB e pilQ mutantes de X. fastidiosa foram encontrados para alterar profundamente a velocidade da contraindo motilidade nas condições fl uxo em micro dispositivos uidic fl 5,6,18. Os estudos realizados sobre a adesão bacteriana e motilidade em dispositivos uidic fl micro indicaram que as câmaras uidic micro fl são particularmente adequadas para analisar a motilidade contrair-se e a migração das bactérias mediada por pili in vitro. Estes resultados explicam o mecanismo de migração mediada por espasmos que facilita a ligação célula-célula, a agregação e a colonização dentroo anfitrião, eventualmente, levar a infecção sistémica. Pil-Chp operon de X. fastidiosa contém pilG, Pili, pilJ, Comprimido, CHPB e CHPC que a transdução do sinal de codificação vias 20. Os quimiorreceptores transmembranares ligar estímulos químicos no domínio periplasmático e activar uma cascata de sinalização na sua porção citoplasmática para controlar, em última análise da motilidade espasmos bacteriana. No operão Pil-cogeração de X. fastidiosa, uma PilG proteína fosfo-shuttle é um homólogo de CheY. Em E. coli e P. aeruginosa, CheY é o regulador de resposta de quimiotaxia em sistemas que interagem com as proteínas do motor flagelos 19, 21. Embora as contribuições do operon Pil-Chp em ​​direção a virulência em X. fastidiosa foram examinados 20 recentemente, o papel de pilG no operão quimiotaxia em resposta aos sinais ambientais e regulada pili de tipo IV / motor de X. fastidiosa é UNClear. Para elucidar a visão do regulador de quimiotaxia pilG na actividade da motilidade espasmos de X. fastidiosa, uma câmara de micro uidic FL é utilizado para avaliar a motilidade espasmos dos X. fastidiosa. O pilG de X. fastidiosa é caracterizada pela comparação dos fenótipos de um mutante de deleção Xf ΔpliG, a estirpe complementar XfΔpliG -C e seu tipo selvagem in vitro. Os resultados destacam o papel de pilG na motilidade espasmos de X. fastidiosa.

<table>
	<tbody>
		<tr>
			<td>Biology materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X. fastidiosa (Xf) Temecula wild type</td>
			<td></td>
			<td></td>
			<td>Costa, H. S., et al., 2004 22</td>
		</tr>
		<tr>
			<td>pilG deletion mutant Xf&#916;pliG</td>
			<td></td>
			<td></td>
			<td>Shi, X. Y., et al., 2007 26</td>
		</tr>
		<tr>
			<td>pilG complementary strain Xf&#916;pliG-C&#160;</td>
			<td></td>
			<td></td>
			<td>Davis, M. J., wt al. 1998 23</td>
		</tr>
		<tr>
			<td>Physical materials and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculating loops</td>
			<td>VWR international, Radnor, PA</td>
			<td>#22-363-607</td>
			<td>quantitative procedures such as bacterial collection</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>#0002709226</td>
			<td>Sylgard 184 silicone Elastomeric Kits</td>
		</tr>
		<tr>
			<td>AmScope MD2000 digital camera</td>
			<td>AmScope, Irvine, CA</td>
			<td>SE305R-AZ-E</td>
			<td>Image, video recording and measurement&#160;</td>
		</tr>
		<tr>
			<td>Tubes line</td>
			<td>Edgewood, NY</td>
			<td>#T4300</td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Plastic luer connectors</td>
			<td>Edgewood, NY</td>
			<td></td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Syringe pumps</td>
			<td>Pico Plus, Harvard Apparatus, MA</td>
			<td>#702209</td>
			<td>The flow rate can be adjusted while the pump is running.</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Gastight, Hemilton Company, Reno, NV</td>
			<td>#1005</td>
			<td>Provide the flowing broth</td>
		</tr>
		<tr>
			<td>Inverted Olympus IMT-2 microscope</td>
			<td>Olympus</td>
			<td>IMT-2 FLuoro PHase</td>
			<td>Image observation and recording</td>
		</tr>
		<tr>
			<td>SPOT-RT digital camera</td>
			<td>Diagnostic Instruments, Inc., MI</td>
			<td>RT230</td>
			<td>Image, video recording and measurement</td>
		</tr>
		<tr>
			<td>Microscope Shutter</td>
			<td>The UNIBLITZ, US</td>
			<td>#LS2T2</td>
			<td>Control camera&#8217;s exposure time</td>
		</tr>
		<tr>
			<td>Microscope Shutter Control system</td>
			<td>The UNIBLITZ, US</td>
			<td>VCM-D1</td>
			<td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td>
		</tr>
		<tr>
			<td>MetaMorph Image software</td>
			<td>Universal Imaging Corp., PA</td>
			<td></td>
			<td>Real-time super-resolution image processing&#160;</td>
		</tr>
	</tbody>
</table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

Xylella fastidiosa é uma bactéria não-flagelado Gram negativo que causa uma série de doenças economicamente importantes de plantas. A motilidade espasmos fornece X. fastidiosa um meio para o movimento de longa distância intra-planta ea colonização, contribuindo para patogenicidade em X. fastidiosa. A motilidade espasmos de X. fastidiosa é operado por pili tipo IV. Pili de tipo IV de Xylella fastidiosa são regulados por pilG, um regulador de quimiotaxia em proteínas do operão de codificação Pil-CHP que estão envolvidos com vias de transdução de sinal. Para elucidar os papéis de pilG na motilidade espasmos de X. fastidiosa, uma pilG -deficient mutante Xf ΔpilG e sua estirpe complementar XfΔpilG- C contendo pilG nativa foram desenvolvidos. A câmara microfluídicos integrados com um sistema de gravação de imagens de lapso de tempo foi usado para observar espasmos motilidade em XfΔpIlg, XfΔpilG- C e sua linhagem selvagem. Usando este sistema de gravação, que permite observações espaciais e temporais de longa duração de agregação, a migração de células individuais e as populações de bactérias através de motilidade espasmos X.. fastidiosa tipo selvagem e complementar estirpe XfΔpilG- C mostrou características típicas espasmos motilidade diretamente observados nas câmaras de fluxo microfluídicos, enquanto mutante XfΔpliG exibiu o fenótipo deficiente se contraindo. Este estudo demonstra que pilG contribui para a motilidade espasmos dos X. fastidiosa. A câmara de fluxo microfluidico é utilizado como um meio para a observação de contracções motilidade.

A presença de uma colónia franja periférica indicativo de tipo IV mediada pelo pilus motilidade espasmos, foi observada nas colónias de X. fastidiosa tipo selvagem e complementar estirpe Xf ΔpliG -C (Figura 1). Mutante XfΔpliG, no entanto, não exibiram uma franja ao redor da periferia de colónias (Figura 1). Imagens de lapso de tempo de células bacterianas em câmaras de fluxo nano-microfluídico revelou que c...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Neste estudo, uma câmara de fluxo nano-microfluídico foi utilizado para visualizar e funcionalmente caracterizar a motilidade espasmos da Xylella fastidiosa, bactéria que causa a doença de Pierce em videira.

Visualização de Twitching motilidade e Caracterização do papel da<em&gt; PilG</em&gt; in<em&gt; Xylella fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells and populations.Demonstrating this procedure will be Dr.Xiangyang Shi, a post-doc from my laboratory.Using a standardized deletion strategy, grow Temecula wild-type Xylella fastidiosa, grow pilG deletion mutants, and grow the complimentary strain all on PD2 medium agar plates.Incubate the bacteria at 28

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Research

JoVE Journal

Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

10.6K visualizações.  University of California, Davis. The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells ...

Vídeo: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Analisar o papel dos Nasopharyngeal-associado do tecido linforreticular (NALT) nas respostas do rato para Vacinas

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

Imunologia e Infecção

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Uma avaliação quantitativa da migração celular pelo ensaio Phagokinetic faixa Motilidade

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Qualitativos e quantitativos Os ensaios para a detecção e caracterização de antimicrobianos Proteína

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Um método rápido para processamento de sangue para aumentar o rendimento dos níveis plasmáticos de peptídeos no sangue humano

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Ensaios para estudar o papel do vitronectina na adesão bacteriana e resistência de soro

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolamento, caracterização e purificação dos macrófagos dos tecidos afetados pela inflamação obesidade

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Isolamento e caracterização de vesículas extracelulares produzidas por micobactérias limitadas por ferro

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Visualização, Quantificação e Mapeamento de Populações de Células Imunes no Microambiente tumoral

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

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 ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Caracterização de Células Imunes e Mediadores Proinflamatórios no Ambiente Pulmonar

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...