1. Die Infektion von Makrophagen mit H. capsulatum, C. albicans <ol><li> 16 Stunden vor der Infektion, Samen Makrophagen an gewünschten Dichte in 24-Well-Platten. In diesem Protokoll wurde eine Dichte von 3,0 x 10 5 Zellen / Vertiefung verwendet. </li><li> In Pilzzellen in log-Wachstumsphase bei einer gewünschten Multiplizität der Infektion (MOI). Dieses Protokoll bietet Platz für eine Reihe von MOI (0,2-5,0). Für die Infektion von Makrophagen mit RAW264.7 H. capsulatum, verwendeten wir eine MOI von 0,2. </li><li> Nach 1,5 Stunden für Phagozytose erlauben, waschen Makrophagen dreimal mit PBS an extrazelluläre Pilze zu entfernen. </li><li> Inkubieren gewünschte Anzahl von Stunden vor Probenanalyse. Bei niedrigen MOI (0.2 in unserem Experiment), bleiben infizierten Makrophagen für mehrere Tage lebensfähig, und die Proben können etwa alle 12-24 Stunden analysiert werden. </li></ol> 2. Makrophagen Lysis <ol><li> Um Pilze aus Makrophagen befreien, entfernen Medien, 3 x waschen mit PBS, und0,5 ml sterilem Wasser. Unter diesen Bedingungen wird Makrophagen lysieren und Pilzzellen bleiben erhalten. </li><li> Inkubieren für 5 min bei Raumtemperatur gerührt. </li><li> Übertragen Lysat sterilen Röhrchen, halten auf dem Eis. </li><li> Übertragen Sie 20 ul Lysat zu einem separaten Röhrchen, dann fügen Sie 20 ul AO / PI-Lösung. Gehen Sie direkt zu Schritt 5: &quot;Probenvorbereitung für Bild Cytometric Analysis&quot; </li></ol> 3. CFU Plating <ol><li> Führen zehnfache Verdünnung der Lysate (aus Schritt 2.3) in den Medien. </li><li> Platte 100 ul jeder Verdünnung in zweifacher Ausfertigung auf HMM-Agaroseplatten. Die Platten in einer feuchten Kammer bei 37 ° C mit 5% CO 2 für 7-8 Tage. </li><li> Manuelles zählen Kolonien auf Platten Darstellung von mindestens 100 und maximal 1000 verschiedene Kolonien. </li></ol> 4. Visualization of Fungi innerhalb Live-Makrophagen <ol><li> Um Live-Makrophagen sammeln, waschen Sie die Zellen 3 mal mit PBS. 0,5 ml PBS und Inkubation für 30 min bei 4 ° C.</li><li> Um Makrophagen aus Gewebekulturvertiefungen entfernen, sanft und Abpipettieren mehrmals. Übertragen Flüssigkeit sterilen Röhrchen, halten auf dem Eis. </li><li> Übertragen Sie 20 ul Probe auf einem separaten Röhrchen, dann fügen Sie 20 ul AO / PI-Lösung. Gehen Sie direkt zu Schritt 5: &quot;Probenvorbereitung für Bild Cytometric Analysis&quot;. </li></ol> 5. Probenvorbereitung für Bild Cytometric Analysis <ol><li> Pipettieren Sie die Target-Probe gründlich und übertragen dann 20 ul Probe in die Zelle Einweg-Zählkammer. </li><li> Lassen Sie die Zellen in der Kammer für 30 Sekunden absetzen. </li><li> Legen Zählkammer in das Bild cytometer. </li></ol> 6. Cellometer Instrument Setup <ol><li> Legen Sie die Fluoreszenzoptik Module: VB-535 bis 402 und VB-660 bis 502 in das System und sicherstellen, dass sie an Ort und Stelle sind gesperrt. <ol><li> VB-535 bis 402 (Anregung bei 475 nm, Emission bei 535 nm) ist für Acridinorange und GFP-Erkennung verwendet. </li><li> VB-660 bis 502 (excitatiauf bei 540 nm wird die Emission bei 660 nm) für Propidiumiodid Detektion verwendet. </li></ol></li><li> Schalten Sie auf das Bild cytometer und öffnen die zugehörige Software. </li></ol> 7. Cellometer Software Setup <ol><li> Wählen Sie die Vorgabe &quot;Assay Type&quot; und &quot;Cell Type&quot; in der Assay Dropdown-Menü. <ol><li> Für Lebensfähigkeit wird der Assay für Acridinorange und Propidiumiodid Erkennung optimiert. </li><li> Für den Nachweis von Candida albicans-Infektion wird der Assay für GFP Erkennung optimiert. </li></ol></li><li> Wählen Sie &quot;Optionen&quot; auf der Oberseite und klicken Sie auf &quot;Take Background Image&quot;, und lassen Sie den Vorgang abgeschlossen ist. </li><li> Klicken Sie auf &quot;Vorschau Hellfeldbild&quot;. </li></ol> 8. Image Acquisition Vorgehensweise <ol><li> Legen Sie die vorbereiteten Probe Kammer in das Bild cytometer. </li><li> Verwenden Sie den Fokus Knopf und stellen Sie den Fokus. </li><li> Einmal im Fokus, auf &quot;Count&quot; klicken, und lassen Sie die Bildaufnahme Vorgang abgeschlossen ist. </li><li> Remove die Einweg-Zählkammer und entsorgen. </li></ol> 9. Bild Datenanalyse <ol><li> Konzentration und Lebensfähigkeit Messung <ol><li> Sobald die Zählung abgeschlossen ist, werden die Konzentration und die Lebensfähigkeit der Zielzellen in der Ergebnis-Seite angezeigt. </li><li> Klicken Sie auf &quot;Export&quot;, um die Daten in FCS Express 4 exportieren für Zellpopulation Analyse der GFP in der Candida albicans-Infektion Experiment. </li></ol></li><li> FCS Express Analyse von Candida albicans-infizierten Zellen <ol><li> Importieren Sie die &quot;. NXDAT&quot; Datei in FCS Express 4 und dann das Ergebnis in einem Fluoreszenz-Histogramm. </li><li> Bewerben Zellpopulation Tor zum Histogramm, um die Bevölkerung Prozentsätze der Candida albicans-infizierten Zellen zu bestimmen. </li></ol></li></ol>

<ol>
	<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. R., Patton, W., Lai, N., Chan, E., Kuksin, D., Lin, B., Qiu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+image-based+cytometry+method+for+autophagy+detection+in+living+cells.">A novel image-based cytometry method for autophagy detection in living cells.</a> Autophagy. 8, 1371-1382 (2012).</li><li>Chan, L. L., Wilkinson, A. R., Paradis, B. D., Lai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Image-based+Cytometry+for+Comparison+of+Fluorescent+Viability+Staining+Methods.">Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods.</a> Journal of Fluorescence. 22, 1301-1311 (2012).</li><li>Chan, L. 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>

Die Autoren Leo Li-Ying Chan und Benjamin Paradis sind Mitarbeiter von Nexcelom Bioscience LLC.

Bildzytometrie ermöglicht es dem Benutzer, um qualitativ hochwertige Bilder zu erfassen und mit spezieller Software, führen Sie eine schnelle Quantifizierung von Zellen. Eine mögliche Herausforderung für die Annahme Bildzytometrie im Bereich der mikrobiellen Pathogenese ist, dass die Mikroorganismen zu zählen, die in einer gemischten Population von Zellen, einschließlich Säuger-Wirtszellen sind. Hier zeigen wir, dass Bildzytometrie zur Quantifizierung von lebensfähigen pathogenen Hefen während der in vitro-...

Studien von pathogenen Pilzen in Verbindung mit ihren Gastgebern und / oder Wirtszellen erfordern oft Quantifizierung der lebensfähigen Pilzzellen über einen Zeitverlauf oder unter verschiedenen Bedingungen Infektion. Zählung der koloniebildenden Einheiten (CFU) der Standard-Methode, durch die die Zahl der lebensfähigen Pilzzellen gemessen wurde, jedoch hat dieses Verfahren einige Nachteile und Einschränkungen. Erstens sind viele Pilzarten langsam wachsende. Das Wachstum der sichtbaren Kolonien auf festen Medien kann 1-2 Wochen, deutlich verlangsamt das Tempo der Forschung. Zweitens ist Manipulation von Proben während der CFU Beschichtung ein langwieriger Prozess, da mehrere Verdünnungen müssen beschichtet, um eine zählbare Anzahl der Kolonien zu gewährleisten. Drittens, ist die Anzahl der CFU typischerweise geringer als die Zahl von lebensfähigen Organismen, weil die Beschichtung überzogen Wirkungsgrad deutlich unter 100%. Zum Beispiel kann Plattieren Wirkungsgrade für die dimorphic Pilzpathogen Histoplasma capsulatum so hoch wie 90%, aber routinemäßig so niedrig wie30% und sogar noch niedriger (10%) für die entsprechende Pilz dimorph Paracoccidioides brasiliensis 1, 2. Plating Effizienz für Candida albicans ist auch Gegenstand Variabilität 3. Schließlich entfallen CFU Analyse für nur leben und sich aktiv teilenden Zellen, die zur Festlegung Wachstum auf festen Medien, wobei in vielen Situationen, wäre es nützlich, um die Anwesenheit und Konzentration von toten und / oder metabolisch inaktiven Zellen zu bestimmen. Zuvor hat durchflusszytometrischen Methoden zur Quantifizierung von mehreren pathogenen Pilzarten wurden 4-6 beschrieben. Doch aufgrund Biosicherheit Containment Probleme bei der Verwendung Sicherheitsstufe 2 (BSL2) oder BSL3-Level Krankheitserreger auf gemeinsamen Durchflusszytometern beteiligt hat Annahme dieser Technik beschränkt. Wie Durchflusszytometrie ist Bildzytometrie eine sensible und schnelle Methode der Zelle Quantifizierung. Allerdings kann Bildzytometrie zu einem Bruchteil der Kosten mit vergleichbaren Ergebnissen 7 durchgeführt werden -11. Hier beschreiben wir Verfahren zur Durchführung Bildzytometrie von pathogenen Pilzen in Verbindung mit Wirtszellen. Wir demonstrieren unsere Methoden mit zwei menschlichen Pilzpathogene: Histoplasma capsulatum und Candida albicans H.. capsulatum ist ein dimorphic Pilzpathogen, die Erkrankung der Atemwege verursacht, in den Menschen, es wächst wie Bäckerhefe und repliziert in Alveolarmakrophagen Candida albicans ist ein Mensch kommensalen Arten, die gelegentlich verursacht Candidiasis.. Wir zeigen, dass Bildzytometrie schnelle Quantifizierung dieser Hefen ermöglicht, zusammen mit Visualisierungsmöglichkeiten.

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			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
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		 <tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000044</td>
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			<td>AO/PI Solution</td>
			<td>Nexcelom Bioscience</td>
			<td>CSK-0102</td>
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			<td>Disposable Counting Chamber</td>
			<td>Nexcelom Bioscience</td>
			<td>CHT4-SD100 </td>
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			<td>EQUIPMENT</td>
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			<td>Cellometer Vision</td>
			<td>Nexcelom Bioscience</td>
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			<td>Cellometer Vision Software</td>
			<td>Nexcelom Bioscience</td>
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use of image cytometry for quantification of pathogenic fungi in association with host cells

Untersuchungen der zellulären Pathogenese pathogener Hefen wie Candida albicans, Histoplasma capsulatum und Cryptococcus neoformans verwenden üblicherweise Infektion von Säugetier-Hosts oder Wirtszellen (zB Makrophagen) durch Hefe Quantifizierung mittels koloniebildende Einheit Analyse oder Durchflusszytometrie gefolgt. Während koloniebildende Einheit Aufzählung ist das am häufigsten verwendete Verfahren auf dem Gebiet hat diese Technik Nachteile und Beschränkungen, einschließlich der langsamen Wachstum einiger Pilzarten auf festen Medien und nieder-und / oder variable Beschichtung Wirkungsgrade, die von besonderer Bedeutung ist bei einem Vergleich Wachstum von Wildtyp-und Mutanten. Durchflusszytometrie können schnelle quantitative Informationen über Hefelebensfähigkeit bieten hat jedoch Verabschiedung durchflusszytometrischen Nachweis für pathogene Hefen für eine Reihe von praktischen Gründen, einschließlich seiner hohen Kosten und die biologische Sicherheit Überlegungen beschränkt. Hier zeigen wir eine bildbasiertecytometric Methodik mit der Cellometer Vision (Nexcelom Bioscience, LLC) für die Quantifizierung von lebensfähigen pathogenen Hefen in Co-Kultur mit Makrophagen. Histoplasma capsulatum und Candida albicans H.:. Unsere Studien auf dem Nachweis von zwei menschlichen Pilzpathogene konzentrieren capsulatum kolonisiert Alveolarmakrophagen durch die Replikation innerhalb der Makrophagen phagosome, und hier haben wir quantitativ das Wachstum von H. capsulatum Hefen in RAW 264.7 Makrophagen mit Acridinorange / Propidiumiodidfärbung in Kombination mit Bildzytometrie. Unsere Methode treu rekapituliert Wachstumstrends wie durch traditionelle Kolonie bildende Einheit Aufzählung gemessen, aber mit deutlich erhöhter Empfindlichkeit. Zusätzlich haben wir direkt beurteilen Infektion von lebenden Makrophagen mit einem GFP-exprimierenden Stamm von C. albicans. Unsere Methodik bietet eine schnelle, genaue und kostengünstige Mittel zur Detektion und Quantifizierung von wichtigen menschlichen pilzliche Erreger in Verbindung with Wirtszellen.

Wir nutzten die Cellometer Bild Anblick cytometer um das Wachstum von H. überwachen capsulatum in Makrophagen. RAW 264.7 Zellen wurden mit H. infiziert capsulatum Hefezellen und zu verschiedenen Zeitpunkten wurden Proben AO / PI-Färbung von bildbasierten durchflusszytometrische Analyse, unterzogen. Parallel dazu wurden Proben von traditionellen CFU Aufzählung analysiert. Zu jedem Zeitpunkt wurden die Proben in Wasser inkubiert, um Makrophagen zu lysieren und die freigesetzte Hefeze...

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

Hier zeigen wir, wie Bildzytometrie zur Quantifizierung von pathogenen Pilzen in Verbindung mit Wirtszellen in der Kultur verwendet werden. Diese Technik kann als Alternative zu CFU Aufzählung verwendet werden.

Verwendung Bildzytometrie zur Quantifizierung von pathogenen Pilzen in Verbindung mit Wirtszellen

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 Aufrufe.  Merrimack College. Hier zeigen wir, wie Bildzytometrie zur Quantifizierung von pathogenen Pilzen in Verbindung mit Wirtszellen in der Kultur verwendet werden. Diese Technik kann als Alternative zu CFU Aufzählung verwendet werden.

Serie: 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.

Quantitative Beurteilung der Immunzellen in das verletzte Rückenmark Tissue mittels Durchflusszytometrie: eine neue Verwendung für eine Zelle Reinigungsverfahren

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

Forschung

Immunologie und Infektion

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.

Die Verwendung einer optischen Falle für das Studium der Wirt-Pathogen-Interaktionen für 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 ...

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.

Die Nutzung der EpiAirway Modell zur Charakterisierung Langfristige Host-Pathogen Interactions

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.

Fluoreszenz-Mikroskopie-Methoden zur Bestimmung der Lebensfähigkeit der Bakterien in Verbindung mit Säugerzellen

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 als Modellorganismus für die Beurteilung der Interaktion von anaeroben Bakterien mit Wirtszellen

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

Die Verwendung von Durchflusszytometrie, um den Zustand von Chromatin in T-Zellen bewerten

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.

Multicolor Flow Cytometry-basierte Quantifizierung der Mitochondrien, Lysosomen in T-Zellen

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.

Ein fortgeschrittenes Murine-Modell für alkoholfreie Steatohepatitis in Verbindung mit Typ-2-Diabetes

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.

Analyse der SAMHD1-Restriktion durch Durchflusszytometrie in humanen myeloischen U937-Zellen

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.

Ein automatisiertes Kultursystem zur präklinischen Erprobung wirtsgerichteter Therapien für Tuberkulose

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