1. Infecção de macrófagos com H. capsulatum, Candida albicans <ol><li> 16 horas antes da infecção, os macrófagos de sementes com uma densidade desejada em placas de 24 poços. Neste protocolo, foi utilizada uma densidade de 3,0 x 10 5 células / poço. </li><li> Adicionar células fúngicas em crescimento de fase log a uma multiplicidade desejada de infecção (MOI). Este protocolo pode acomodar uma gama de MOI (0,2-5,0). Para a infecção de macrófagos RAW264.7 com H. capsulatum, foi utilizada uma MOI de 0,2. </li><li> Após 1,5 horas para permitir a fagocitose, macrófagos lavar três vezes com PBS para remover fungos extracelulares. </li><li> Incubar número desejado de horas antes da análise de amostras. Em baixa MOI (0,2 na nossa experiência), células de macrófagos infectados permanecem viáveis ​​durante vários dias, e as amostras podem ser analisadas aproximadamente a cada 12-24 horas. </li></ol> 2. Lise de macrófagos <ol><li> Para libertar os fungos a partir de células de macrófagos, ao remover a mídia, lavar 3 vezes com PBS, eadicionar 0,5 ml de água estéril. Sob estas condições, os macrófagos e irá lisar células fúngicas permanecerá intacta. </li><li> Incubar durante 5 min a temperatura ambiente. </li><li> Transferir lysate para tubo estéril, manter em gelo. </li><li> Transferir 20 uL de ligado a um tubo separado, em seguida, adicionar 20 ul AO / PI solução. Prosseguir diretamente para a etapa 5: &quot;Preparação de Amostras para análise por citometria de imagem&quot; </li></ol> 3. UFC chapeamento <ol><li> Realizar diluição de dez vezes de lisados ​​(do passo 2.3) em meios. </li><li> Placa de 100 ul de cada diluição em duplicado, em placas HMM-agarose. Incubar as placas numa câmara húmida a 37 ° C com 5% de CO 2 durante 7-8 dias. </li><li> Contar manualmente colônias em placas indicam um mínimo de 100 e um máximo de 1.000 colônias distintas. </li></ol> 4. Visualização de fungos no interior dos macrófagos ao vivo <ol><li> Para a coleta de macrófagos ao vivo, lave as células três vezes com PBS. Adicionar 0,5 ml de PBS, e incubar durante 30 min a 4 ° C.</li><li> Para remover os macrófagos dos poços de cultura de tecidos, pipeta suavemente para cima e para baixo várias vezes. Transferência líquida para tubo estéril, manter em gelo. </li><li> Transferir 20 uL de amostra a um tubo separado, em seguida, adicionar 20 ul AO / PI solução. Prosseguir diretamente para a etapa 5: &quot;Preparação de Amostras para análise por citometria de imagem&quot;. </li></ol> 5. Preparação de amostras para análise por citometria de imagem <ol><li> Pipetar a amostra-alvo e completamente em seguida, transferir 20 mL de amostra para a célula descartável câmara de contagem. </li><li> Permitir que as células se instalar na câmara por 30 segundos. </li><li> Insira câmara de contagem para o citômetro de imagem. </li></ol> 6. Configuração do Instrumento Cellometer <ol><li> Insira a ótica de fluorescência Módulos: VB-535-402 e VB-660-502 para o sistema e verifique se eles estão presos no local. <ol><li> VB-535-402 (excitação a 475 nm, emissão a 535 nm), é usada para a detecção de laranja de acridina e de GFP. </li><li> VB-660-502 (excitatiem 540 nm, emissão a 660 nm) é utilizado para a detecção de iodeto de propídio. </li></ol></li><li> Ligue o citômetro de imagem e abrir o software que o acompanha. </li></ol> 7. Instalação de Software Cellometer <ol><li> Selecione o preset &quot;Tipo de Ensaio&quot; e &quot;Tipo de célula&quot; no menu suspenso de Ensaio. <ol><li> Para a viabilidade, o ensaio é optimizado para a detecção de laranja de acridina e iodeto de propídio. </li><li> Para a detecção de infecção por Cândida albicans, o ensaio é optimizado para a detecção de GFP. </li></ol></li><li> Selecione &quot;Opções&quot; na parte superior e clique em &quot;Take imagem de fundo&quot;, e permitir que a operação seja concluída. </li><li> Clique em &quot;Imagem de campo claro.&quot; </li></ol> 8. Imagem Procedimento de Aquisição <ol><li> Insira na câmara de amostra preparada para o citômetro de imagem. </li><li> Use o botão de foco e ajustar o foco. </li><li> De vez em foco, clique em &quot;Count&quot;, e permitir que a operação de aquisição de imagem para ser concluído. </li><li> Remove a câmara de contagem descartáveis ​​e descartar adequadamente. </li></ol> 9. Imagem de Análise de Dados <ol><li> Concentração e de medição da viabilidade <ol><li> Uma vez que a contagem é concluída, a concentração e a viabilidade das células-alvo são apresentados na página de resultado. </li><li> Clique em &quot;Exportar&quot; para exportar os dados para o FCS Express 4 para análise da população celular da GFP em Candida albicans experiência infecção. </li></ol></li><li> Análise FCS expresso de células infectadas com Cândida albicans <ol><li> Importe o arquivo &quot;. NXDAT&quot; em FCS Express 4 e enredo os resultados em um histograma de fluorescência. </li><li> Aplicar portão população de células com o histograma para determinar a percentagem de população de células infectadas com Cândida albicans. </li></ol></li></ol>

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	<li>Worsham, P. L., Goldman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+plating+of+Histoplasma+capsulatum+without+addition+of+conditioned+medium+or+siderophores.">Quantitative plating of Histoplasma capsulatum without addition of conditioned medium or siderophores.</a> J. Med. Vet. Mycol. 26, 137-143 (1988).</li><li>Goihman-Yahr, M., Pine, L., Albornoz, M. C., Yarzabal, L., de Gomez, M. H., San Martin, B., Ocanto, A., Molina, T., Convit, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells.">Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.</a> Mycopathologia. 71, 73-83 (1980).</li><li>Bhatti, M. A., Hjertstedt, J., Hahn, B. L., Sohnle, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+delivery+of+yeast+cells+as+an+explanation+for+reduced+plating+efficiency+of+Candida+albicans.">Inefficient delivery of yeast cells as an explanation for reduced plating efficiency of Candida albicans.</a> Med. Mycol. 40, 465-469 (2002).</li><li>Chang, W. L., vander Heyde, H. C., Klein, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+quantitation+of+yeast:+a+novel+technique+for+use+in+animal+model+work+and+in+vitro+immunologic+assays.">Flow cytometric quantitation of yeast: a novel technique for use in animal model work and in vitro immunologic assays.</a> Journal of Immunological Methods. 211, 51-63 (1998).</li><li>Green, L., Petersen, B., Steimel, L., Haeber, P., Current, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+antifungal+activity+by+flow+cytometry.">Rapid determination of antifungal activity by flow cytometry.</a> Journal of Clinical Microbiology. 32, 1088-1091 (1994).</li><li>Kirk, S. M., Callister, S. M., Lim, L. C., Schell, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+susceptibility+testing+of+Candida+albicans+by+flow+cytometry.">Rapid susceptibility testing of Candida albicans by flow cytometry.</a> Journal of Clinical Microbiology. 35, 358-363 (1997).</li><li>Chan, L. L. -. Y., Lai, N., Wang, E., Smith, T., Yang, X., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+detection+method+for+apoptosis+and+necrosis+measurement+using+the+Cellometer+imaging+cytometry.">A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry.</a> Apoptosis. 16, 1295-1303 (2011).</li><li>Chan, L. L. -. Y., Shen, D., Wilkinson, A. 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L., Zhong, X., Pirani, A., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+kinetic+measurements+of+rare+cell+proliferation+using+Cellometer+image-based+cytometry.">A novel method for kinetic measurements of rare cell proliferation using Cellometer image-based cytometry.</a> J. Immunol. Methods. 377, 8-14 (2012).</li><li>Chan, L. L., Zhong, X., Qiu, J., Li, P. Y., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellometer+Vision+as+an+alternative+to+flow+cytometry+for+cell+cycle+analysis,+mitochondrial+potential,+and+immunophenotyping.">Cellometer Vision as an alternative to flow cytometry for cell cycle analysis, mitochondrial potential, and immunophenotyping.</a> Cytom. Part A. 79, 507-517 (2011).</li><li>Hull, C. M., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mating+type-like+locus+in+the+asexual+pathogenic+yeast+Candida+albicans.">Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans.</a> Science. 285, 1271-1275 (1999).</li><li>Berkes, C. A., Chan, L. L., Wilkinson, A., Paradis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Quantification+of+Pathogenic+Fungi+by+Cellometer+Image-Based+Cytometry.">Rapid Quantification of Pathogenic Fungi by Cellometer Image-Based Cytometry.</a> J. Micro. Meth. 91, 468-476 (2012).</li><li>Nguyen, V. Q., Sil, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-induced+switch+to+the+pathogenic+yeast+form+of+Histoplasma+capsulatum+requires+Ryp1,+a+conserved+transcriptional+regulator.">Temperature-induced switch to the pathogenic yeast form of Histoplasma capsulatum requires Ryp1, a conserved transcriptional regulator.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (12), 4880-4885 (2008).</li><li>Wenisch, C., Linnau, K. F., Parschalk, B., Zedtwitz-Liebenstein, K., Georgopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+susceptibility+testing+of+fungi+by+flow+cytometry+using+vital+staining.">Rapid susceptibility testing of fungi by flow cytometry using vital staining.</a> Journal of Clinical Microbiology. 35, 5-10 (1997).</li></ol>

Os autores Leo Li-Ying Chan e Benjamin Paradis são funcionários da Nexcelom Bioscience LLC.

Citometria de imagem permite ao usuário capturar imagens de alta qualidade e, usando um software especializado, realizar rápida quantificação de células. Um desafio potencial para a adopção de citometria de imagem no domínio da patogênese microbiana é que os microrganismos sejam contados presente numa população mista de células, incluindo células hospedeiras de mamífero. Aqui, demonstramos que a citometria de imagem pode ser utilizada para a quantificação de leveduras patogénicas viáveis ​​durante...

Estudos de fungos patogénicos em associação com os seus hospedeiros e / ou células hospedeiras exigem frequentemente a quantificação de células de fungos viáveis ​​ao longo de um curso de tempo ou sob diferentes condições de infecção. Enumeração das unidades formadoras de colónias (UFC) é o método padrão pelo qual o número de células de fungos viáveis ​​foi medida, no entanto, esta técnica tem várias desvantagens e limitações. Em primeiro lugar, muitas espécies de fungos são de crescimento lento. O crescimento das colônias visíveis em meios sólidos pode levar 1-2 semanas, diminuindo significativamente o ritmo da investigação. Por outro lado, a manipulação de amostras durante CFU chapeamento é um processo laborioso, uma vez que várias diluições deve ser revestida para garantir um número de colónias contáveis. Em terceiro lugar, o número de UFC é tipicamente menor do que o número de organismos viáveis ​​plaqueadas porque a eficiência de plaqueamento é bem abaixo de 100%. Por exemplo, a eficiência de revestimento para o dimórfico Histoplasma capsulatum patógeno fúngico pode ser tão alto como 90%, mas são habitualmente tão acessível quanto30% e são ainda mais baixos (10%) para o fungo relacionado Dimorfa Paracoccidioides brasiliensis 1, 2. Eficiência chapeamento de Candida albicans também está sujeito à variabilidade 3. Finalmente, a análise de CFU representa apenas as células vivas e dividindo activamente capazes de estabelecer o crescimento em meios sólidos, ao passo que, em muitas situações, pode ser útil para determinar a presença e concentração de células mortas inactivos e / ou metabolicamente. Anteriormente, os métodos de citometria de fluxo para a quantificação de várias espécies de fungos patogénicos tem sido descrita 4-6. No entanto, devido a questões de biossegurança de contenção envolvidos com o uso de nível de biossegurança 2 (BSL2) ou patógenos de nível BSL3 em cytometers fluxo compartilhados, a adoção dessa técnica tem sido limitada. Como citometria de fluxo, citometria de imagem é um método sensível e rápido de quantificação celular. No entanto, a imagem de citometria pode ser realizada a uma fracção do custo com resultados comparáveis ​​7 -11. Aqui, descrevemos os métodos para a realização de citometria de imagem de fungos patogênicos em associação com células hospedeiras. Nós demonstramos nossos métodos usando dois fungos patógenos humanos: Histoplasma capsulatum e Candida albicans H.. capsulatum é um fungo dimórfico que causa doença respiratória, em seres humanos, que cresce levedura como brotamento e replica no interior de macrófagos alveolares Candida albicans é uma espécie comensais humanos que provoca ocasionalmente candidíase.. Nós mostramos que a citometria de imagem permite uma rápida quantificação dessas leveduras, juntamente com a capacidade de visualização.

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use of image cytometry for quantification of pathogenic fungi in association with host cells

Estudos dos mecanismos de patogênese celulares de leveduras patogênicas como a Candida albicans, Histoplasma capsulatum e Cryptococcus neoformans comumente empregam infecção de hospedeiros mamíferos ou células hospedeiras (ou seja, macrófagos), seguido pela quantificação de leveduras utilizando análise de unidade formadora de colônia ou citometria de fluxo. Enquanto a unidade de formação de colónia enumeração tem sido o método mais comummente utilizado no campo, esta técnica tem as desvantagens e limitações, incluindo crescimento lento de algumas espécies de fungos em meios sólidos e eficiências de baixo e / ou variável de revestimento, que é de preocupação particular quando se compara o crescimento de estirpes de tipo selvagem e mutante. A citometria de fluxo podem proporcionar informação quantitativa rápida quanto viabilidade celular, no entanto, a adopção de citometria de fluxo para a detecção de leveduras patogénicas tem sido limitada por um número de razões práticas, incluindo o seu elevado custo e as considerações de biossegurança. Aqui, nós demonstramos uma baseada em imagemmetodologia de citometria usando a visão Cellometer (Nexcelom Bioscience, LLC), para a quantificação de leveduras patogênicos em co-cultura com macrófagos. Nossos estudos concentram-se na detecção de dois fungos patógenos humanos: Histoplasma capsulatum e Candida albicans H.. capsulatum coloniza macrófagos alveolares, replicando dentro do fagossoma do macrófago, e aqui, nós quantitativamente avaliar o crescimento de H. leveduras capsulatum em RAW 264,7 macrófagos usando laranja de acridina / coloração iodeto de propídio em combinação com a citometria de imagem. Nosso método fielmente recapitula as tendências de crescimento, medida pela unidade formadora de colônia tradicional enumeração, mas com um aumento significativo de sensibilidade. Além disso, avaliar diretamente a infecção de macrófagos ao vivo com uma cepa GFP-expressando de C. albicans. Nossa metodologia oferece um meio rápido, preciso e econômico para a detecção e quantificação de importantes fungos patógenos humanos em associação sagacidadeAs células hospedeiras h.

Usamos o Cellometer Visão imagem citômetro para monitorar o crescimento de H. capsulatum do macrófago. Células RAW 264.7 foram infectados com H. células de levedura capsulatum e em vários pontos de tempo, as amostras foram submetidas a AO / PI coloração seguida por análise de citometria de imagem baseado. Em paralelo, as amostras foram analisadas por contagem de CFU tradicional. Em cada ponto de tempo, as amostras foram incubadas em água para lisar os macrófagos e as células de lev...

Watch this Scientific Journal Video about Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells at JoVE.com

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Aqui, demonstramos como citometria de imagem pode ser usada para quantificação de fungos patogénicos em associação com as células hospedeiras em cultura. Esta técnica pode ser usado como uma alternativa para CFU enumeração.

Uso de Citometria de imagem para quantificação de fungos patogênicos em associação com células do hospedeiro

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

use-image-cytometry-for-quantification-pathogenic-fungi-association

Research

JoVE Journal

Immunology and Infection

12.8K visualizações.  Merrimack College. Aqui, demonstramos como citometria de imagem pode ser usada para quantificação de fungos patogénicos em associação com as células hospedeiras em cultura. Esta técnica pode ser usado como uma alternativa para CFU enumeração.

Vídeo: Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Avaliação quantitativa das células imunes no tecido medula lesionada por Citometria de Fluxo: um novo uso para um método de purificação celular

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Imunologia e Infecção

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Uso de uma armadilha óptica de Estudos de Interações Hospedeiro-Patógeno para imagens de células dinâmica ao vivo

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Utilização do Modelo EpiAirway para caracterizar a longo prazo Interações Hospedeiro-Patógeno

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

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 ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis como um organismo modelo para a Avaliação da Interação de bactérias anaeróbias com células do hospedeiro

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

A citometria de fluxo para avaliar o estado de cromatina em células T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Quantificação de baseados em citometria de fluxo multicolor de mitocôndrias e lisossomos em células T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Um modelo murino avançado para esteatohepatite não alcoólica em associação com diabetes tipo 2

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Análise da Restrição de SAMHD1 por Citometria de Fluxo em Células U937 Mieloides Humanas

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Um Sistema de Cultura Automatizado para Uso em Testes Pré-Clínicos de Terapias Dirigidas ao Hospedeiro para Tuberculose

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...