Este trabajo está financiado por el Programa de Investigación Investigador Joven, del Centro de Investigación de Enfermedades Infecciosas (Zinf) de la Universidad de Würzburg. Juan C García-Betancur es un compañero de doctorado de la Escuela de Graduados en Ciencias de la Vida (GSL) de la Universidad de Würzburg.

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ica+and+beyond:+biofilm+mechanisms+and+regulation+in+Staphylococcus+epidermidis+and+Staphylococcus+aureus.">ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus.</a> FEMS Microbiol Lett. 270, 179-188 (2007).</li><li>Chai, Y., Chu, F., Kolter, R., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistability+and+biofilm+formation+in+Bacillus+subtilis.">Bistability and biofilm formation in Bacillus subtilis.</a> Mol. Microbiol. 67, 254-263 (2008).</li><li>Chen, R., Guttenplan, S. B., Blair, K. M., Kearns, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+sigmaD-dependent+autolysins+in+Bacillus+subtilis+population+heterogeneity.">Role of the sigmaD-dependent autolysins in Bacillus subtilis population heterogeneity.</a> J. Bacteriol. 191, 5775-5784 (2009).</li><li>Guttenplan, S. B., Blair, K. M., Kearns, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EpsE+flagellar+clutch+is+bifunctional+and+synergizes+with+EPS+biosynthesis+to+promote+Bacillus+subtilis+biofilm+formation.">The EpsE flagellar clutch is bifunctional and synergizes with EPS biosynthesis to promote Bacillus subtilis biofilm formation.</a> PLoS Genet. 6, e1001243-e1001243 (2010).</li><li>Kearns, D. B., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+population+heterogeneity+during+growth+of+Bacillus+subtilis.">Cell population heterogeneity during growth of Bacillus subtilis.</a> Genes Dev. 19, 3083-3094 (2005).</li><li>Lopez, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+signaling+in+a+bacterium.">Paracrine signaling in a bacterium.</a> Genes Dev. 23, 1631-1638 (2009).</li><li>Veening, J. W., Smits, W. K., Hamoen, L. W., Jongbloed, J. D., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+differential+gene+expression+by+improved+cyan+fluorescent+protein+and+yellow+fluorescent+protein+production+in+Bacillus+subtilis.">Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis.</a> Appl. Environ. Microbiol. 70, 6809-6815 (2004).</li><li>Veening, J. W., Smits, W. K., Hamoen, L. W., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+gene+expression+patterns+of+competence+development+and+initiation+of+sporulation+in+Bacillus+subtilis+grown+on+chemically+defined+media.">Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media.</a> J. Appl. Microbiol. 101, 531-541 (2006).</li><li>Veening, J. W., Kuipers, O. P., Brul, S., Hellingwerf, K. J., Kort, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+phosphorelay+perturbations+on+architecture,+sporulation,+and+spore+resistance+in+biofilms+of+Bacillus+subtilis.">Effects of phosphorelay perturbations on architecture, sporulation, and spore resistance in biofilms of Bacillus subtilis.</a> J. Bacteriol. 188, 3099-3109 (2006).</li><li>Vlamakis, H., Aguilar, C., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+fate+by+the+formation+of+an+architecturally+complex+bacterial+community.">Control of cell fate by the formation of an architecturally complex bacterial community.</a> Genes Dev. 22, 945-953 (2008).</li><li>Stewart, P. S., Franklin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+heterogeneity+in+biofilms.">Physiological heterogeneity in biofilms.</a> Nat. Rev. Microbiol. 6, 199-210 (2008).</li><li>Veening, J. W., Smits, W. K., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistability,+epigenetics,+and+bet-hedging+in+bacteria.">Bistability, epigenetics, and bet-hedging in bacteria.</a> Annu. Rev. Microbiol. 62, 193-210 (2008).</li><li>Aguilar, C., Vlamakis, H., Guzman, A., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KinD+is+a+checkpoint+protein+linking+spore+formation+to+extracellular-matrix+production+in+Bacillus+subtilis+biofilms.">KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms.</a> MBio. 1, (2010).</li><li>Lopez, D., Fischbach, M. A., Chu, F., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structurally+diverse+natural+products+that+cause+potassium+leakage+trigger+multicellularity+in+Bacillus+subtilis.">Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 280-285 (2009).</li><li>Lopez, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannibalism+enhances+biofilm+development+in+Bacillus+subtilis.">Cannibalism enhances biofilm development in Bacillus subtilis.</a> Mol. Microbiol. 74, 609-618 (2009).</li><li>Arima, K., Kakinuma, A., Tamura, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactin,+a+crystalline+peptidelipid+surfactant+produced+by+Bacillus+subtilis:+isolation,+characterization+and+its+inhibition+of+fibrin+clot+formation.">Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation.</a> Biochem. Biophys. Res. Commun. 31, 488-494 (1968).</li><li>Romero, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+accessory+protein+required+for+anchoring+and+assembly+of+amyloid+fibres+in+B.+subtilis+biofilms.">An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms.</a> Mol. Microbiol. 80, 1155-1168 (2011).</li><li>Hardwood, C. R., Cutting, S. M. <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 Biological Methods for Bacillus. , (1990).</li><li>Novick, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+systems+in+staphylococci.">Genetic systems in staphylococci.</a> Methods Enzymol. 204, 587-636 (1991).</li><li>Yasbin, R. E., Young, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+in+Bacillus+subtilis+by+bacteriophage+SPP1.">Transduction in Bacillus subtilis by bacteriophage SPP1.</a> J. Virol. 14, 1343-1348 (1974).</li><li>Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruiting+body+formation+by+Bacillus+subtilis.">Fruiting body formation by Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 11621-11626 (2001).</li><li>Nakano, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=srfA+is+an+operon+required+for+surfactin+production,+competence+development,+and+efficient+sporulation+in+Bacillus+subtilis.">srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis.</a> J. Bacteriol. 173, 1770-1778 (1991).</li><li>Gonzalez-Pastor, J. E., Hobbs, E. C., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannibalism+by+sporulating+bacteria.">Cannibalism by sporulating bacteria.</a> Science. 301, 510-513 (2003).</li><li>Aguilar, C., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+Bacillus+subtilis+as+a+multicellular+organism.">Thinking about Bacillus subtilis as a multicellular organism.</a> Curr. Opin. Microbiol. 10, 638-643 (2007).</li><li>Shapiro, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu. Rev. Microbiol. 52, 81-104 (1998).</li></ol>

No tenemos nada que revelar.

El hecho de que las comunidades bacterianas muestran las subpoblaciones de células que expresan el conjunto específico de genes evidencia la complejidad de las comunidades microbianas 33,34. Este protocolo debe ayudar a determinar si la expresión de cualquier gen de interés se limita a una subpoblación particular de células especializadas dentro de la comunidad microbiana. La visualización de estas subpoblaciones requiere el desarrollo de nuevas técnicas, porque los métodos tradicionales para control...

<table border="1"><tbody><tr><td> Técnica </td><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td rowspan="11"> MSgg composición </td><td> 5 mM de fosfato de potasio </td><td> Roth </td><td> 6878 </td></tr><tr><td> MOPS 100 mm </td><td> Sigma-Aldrich </td><td> M1254 </td></tr><tr><td> Cloruro de magnesio 2 mM </td><td> Roth </td><td> 2189.1 </td></tr><tr><td> 700μM de cloruro de calcio </td><td> Roth </td><td> A119.1 </td></tr><tr><td> 50 micras de cloruro férrico </td><td> Sigma-Aldrich </td><td> 157740 </td></tr><tr><td> 1um cloruro de cinc </td><td> Applichem </td><td> A2076 </td></tr><tr><td> La tiamina 2μM </td><td> Sigma-Aldrich </td><td> 74625 </td></tr><tr><td> El glicerol 0,5% </td><td> Roth </td><td> 7533 </td></tr><tr><td> Glutacompañero de 0,5% </td><td> Sigma-Aldrich </td><td> 49621 </td></tr><tr><td> El triptófano 50μg/ml </td><td> Sigma-Aldrich </td><td> T0254 </td></tr><tr><td> La fenilalanina 50μg/ml </td><td> Sigma-Aldrich </td><td> P2126 </td></tr><tr><td> La fijación de la célula </td><td> Paraformaldehído </td><td> Roth </td><td> 0335 </td></tr><tr><td></td><td> Nombre del equipo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Sonicación </td><td> Celular Sonicator </td><td> Bandelin </td><td> D-1000 </td></tr><tr><td> Microscopía de fluorescencia </td><td> Microscopio de fluorescencia </td><td> Leica </td><td> DMI6000B </td></tr><tr><td></td><td> Nombre del software </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Microscopía de fluorescencia </td> &lt;td&gt; Asaf </td><td> Leica </td><td></td></tr><tr><td> La citometría de flujo </td><td> FCASDiva </td><td> BD </td><td></td></tr><tr><td> La citometría de flujo </td><td> FlowJo </td><td> Treestar </td><td></td></tr></tbody></table>

single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry

La formación de biopelículas es un atributo general a casi todas las bacterias 1-6. Cuando las bacterias forman biopelículas, las células están encerradas en una matriz extracelular que está mayoritariamente constituida por las proteínas y exopolisacáridos, entre otros 7-10. La comunidad microbiana encerrado dentro de la biopelícula a menudo muestra la diferenciación de las distintas subpoblaciones de células especializadas 11-17. Estas subpoblaciones coexisten y se muestran a menudo la organización espacial y temporal dentro del biofilm 18-21. La formación de biopelículas en los Bacillus subtilis organismo modelo requiere la diferenciación de las distintas subpoblaciones de células especializadas. Entre ellos, la subpoblación de los productores de matriz, responsables de producir y secretar la matriz extracelular de la biopelícula es esencial para la formación de biopelículas 11,19. Por lo tanto, la diferenciación de los productores de la matriz es una característica de la formación de biopelículas en la B. subtilis. Hemos utilizado los reporteros fluorescentes para visualizar y cuantificar la subpoblación de los productores de la matriz en las biopelículas de B. subtilis 15,19,22-24. Concretamente, hemos observado que la subpoblación de los productores de matriz diferencia en respuesta a la presencia de auto-producido señal extracelular surfactina 25. Curiosamente, surfactina es producida por una subpoblación de células especializadas diferentes de la subpoblación de los productores de matriz 15. Hemos expuesto en este informe, el enfoque técnico necesario para visualizar y cuantificar la subpoblación de los productores de la matriz y los productores de surfactina en las biopelículas de B. subtilis. Para hacer esto, los reporteros fluorescentes de los genes necesarios para la producción de matriz y la producción de surfactina se insertan en el cromosoma de B. subtilis. Los reporteros se expresan únicamente en una subpoblación de células especializadas. Entonces, las subpoblaciones puede sercontrolará mediante microscopía de fluorescencia y citometría de flujo (ver Fig. 1). El hecho de que diferentes subpoblaciones de células especializadas coexisten dentro de las comunidades de bacterias multicelulares nos da una perspectiva diferente sobre la regulación de la expresión génica en procariotas. Este protocolo trata este fenómeno de manera experimental y puede ser fácilmente adaptado a cualquier modelo de otros tratamientos, para dilucidar los mecanismos moleculares que subyacen a la heterogeneidad fenotípica dentro de una comunidad microbiana.

Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biopelículas microbianas están constituidas generalmente por distintas subpoblaciones de células especializadas. Sola célula de análisis de estas subpoblaciones requiere el uso de reporteros fluorescentes. A continuación se describe un protocolo para visualizar y controlar varias subpopulationswithin<em&gt; B. subtilis</em&gt; Biopelículas utilizando microscopía de fluorescencia y citometría de flujo.

Sola célula de análisis de Bacillus subtilis Las biopelículas mediante microscopía de fluorescencia y citometría de flujo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

single-cell-analysis-bacillus-subtilis-biofilms-using-fluorescence

Research

JoVE Journal

Immunology and Infection

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

20.3K vistas.  University of Würzburg. Biopelículas microbianas están constituidas generalmente por distintas subpoblaciones de células especializadas. Sola célula de análisis de estas subpoblaciones requiere el uso de reporteros fluorescentes. A continuación se describe un protocolo para visualizar y controlar varias subpopulationswithin B. subtilis Biopelículas utilizando microscopía de fluorescencia y citometría de flujo.

Video: Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análisis por citometría de flujo de células inmunes dentro de aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Investigación

Inmunología e Infección

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imágenes de células vivas de Bacillus subtilis Y Streptococcus pneumoniae Automatizada utilizando Time-lapse microscopía

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un protocolo simple y rápida para no enzimáticamente disociarse Tejidos Humanos frescas para el Análisis de los linfocitos infiltrantes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Prevención de Estrés Térmico efectos adversos en ratas<em&gt; Bacillus subtilis</em&gt; Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologías para el estudio<em&gt; B. subtilis</em&gt; Las biopelículas como modelo para la caracterización de Pequeño Biofilm inhibidores de moléculas

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterización de subconjuntos de monocitos humanos de sangre análisis de citometría de flujo

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Cuantificación de Efferocytosis por microscopía de fluorescencia unicelular

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparación de la médula ósea entera para el análisis de citometría de masa de células de linaje neutrófilo

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...