Vision-Health Research Network - Diese Arbeit wird durch Zuschüsse aus dem kanadischen Institutes of Health Research (178.918), Fonds de recherche en santé du Québec unterstützt.

HINWEIS: Männliche C57BL / 6-Mäuse (5-7 Wochen alt) werden von Charles River Laboratories International, Inc. (St-Constant, Quebec, Kanada.) Und nach Protokollen von der CHU Sainte-Justine Animal Care Committee genehmigt manipuliert. Maus Bronchialgewebe Explantate eine gute Quelle von Primär bronchialen Epithelzellen zur Untersuchung der proapoptotischen Wirkung von LMPs auf Epithelzellen. Dieses Protokoll beschreibt die synthetische Entwicklung LMPs sowie ein Verfahren zum Nachweis apoptotischer Epithelzellen auf LMPs behandelten bronchiale Gewebeexplantaten. Dieses Protokoll besteht aus drei Abschnitten. 1. LMPs Herstellung und Charakterisierung HINWEIS: Um eine Kontamination zu vermeiden, stellen Sie sicher, dass alle in diesem Experiment verwendeten Materialien sind steril und autoklaviert. Alle Schritte bei Raumtemperatur in einem biologischen Sicherheitsschrank unter sterilen Bedingungen, sofern nicht anders angegeben. 1.1) Stimulation und Sammlung von Abgeordneten9 <ol><li> Auftauen ein Aliquot von 10 Millionen CEM-T-Zellen in einem 37 ° C Wasserbad. Verdünne in 10 ml vorgewärmten serumfreien hämatopoetischen Medium wie X-VIVO, in einem 15 ml sterilen Röhrchen und zentrifugiert bei 200 g × 5 min. Saugen Sie den Überstand und resuspendieren Zellen in 5 ml vorgewärmten Medium. </li><li> Übertragungszellen in einen T75-Gewebekulturkolben (für Suspensionskulturen) mit 15 ml vorgewärmten hämatopoetischen Medium wie X-VIVO und Inkubation für 4 Tage in einem befeuchteten Inkubator bei 37ºC mit 5% CO 2. </li><li> Nach 4 Tagen übertragen alle Kulturmedium und Zellen in einen T175 Gewebekulturflasche mit 100 ml frischem Medium. Weiter Inkubieren der Zellen für ungefähr 72 Stunden unter den gleichen Bedingungen, bis sie zu einer Dichte von 2 Mio Zellen / ml gezüchtet. </li><li> Gleichmäßig auf vier T175-Flaschen, die jeweils 150 ml frischem Medium und weiterhin Zellkultur, bis Zellen (etwa 48 h Inkubation) zu einer Dichte von 2 Millionen / ml gezüchtet Teilen von Zellen. </li><li&gt; Sammeln Zellen aus jedem Kolben durch Zentrifugation bei 200 xg für 5 Minuten und Resuspendieren 300 x 10 6 Zellen in ein neues T175-Kolben, der 150 ml frisches Medium, um den 2 Millionen / ml Zelldichte aufrechtzuerhalten. </li><li> Hinzufügen Actinomycin D (gelöst in DMSO bei 2 mg / ml) zu dem Medium in einer Endkonzentration von 0,5 ug / ml und Inkubation für 24 Std. </li><li> Lassen Sie das gesamte Kulturmedium in 50 ml konischen Röhrchen und Spin-down werden die Zellen bei 750 g für 5 min. Den Überstand in 50 ml konische Röhrchen und zentrifugieren bei 1.500 g für 15 Minuten, um große Zellbruchstücke zu entfernen. </li><li> Den Überstand in einen 250 ml-Flasche und Ultrazentrifuge bei 12.000 × g für 50 min. Überstand verwerfen und sammeln Pellets. </li><li> Wasch LMPs angereicherten Pellets mit 40 ml sterilem PBS in ein 50 ml Röhrchen durch Zentrifugation bei 12.000 × g für 50 min. Wiederholen Sie diesen Schritt zweimal. </li><li> Sammeln Sie die letzten Waschüberstand; wird sie als Fahrzeugsteuerung verwendet werden. Suspend die LMPs Pellets in 1ml PBS und Übertragung in einem 1,5 ml sterilen Mikroröhrchen. Aliquotieren und Speicher isoliert LMPs bei -80 ° C (um mehrere freie und Auftauen zu vermeiden). </li></ol> 1.2) Charakterisierung der Abgeordneten über FACS-Analyse 4 <ol><li> Planen 2 Proben von Annexin Puffer, 1 mit und andere ohne CaCl 2: Hepes 10 mM, NaCl 140 mM, plus oder minus 5 mM CaCl 2. </li><li> Filtern Annexin Puffer und FACS Strömungs Hüllfluid mit einem 0,22 um Filter, um Partikel zu entfernen. </li><li> Verdünnen Sie 1 ul LMPs in 44 ul von Annexin-Puffer mit 5 mM CaCl 2 in einer FACS-Röhrchen. Bereiten anderes Rohr mit 1 ul LMPs in 44 ul Annexin Puffer ohne CaCl 2 (Negativkontrolle). </li><li> In 5 ul der Annexin-Cy5 in jeder Röhre und gut mischen. Inkubieren für 15 min bei RT im Dunkeln. Die Reaktion durch Verdünnen der Mischung mit 400 ul FACS Fluss Mantelfluid in jedem Röhrchen. </li><li> In 10 ul (200.000 Kügelchen) von 7 um das Zählen beads Suspension als interner Standard in jedem Röhrchen, um eine absolute Anzahl ermitteln. </li><li> Stellen Toren der relativen Größe (FSC-H, PMT E00, logarithmischer Maßstab) und die relative Granularität (SSC-H, PMT 325, logarithmischer Maßstab) Dot-Plot auf dem Durchflusszytometer mit Größe kalibriert fluoreszierende Kügelchen von 1 um (Tor 1) und Zählen Perlen Tor bei 7 um (Tor 2). </li><li> Analysieren Sie den LMPs Probe auf FSC-H / SSC-H Grundstück mit den etablierten Toren und FL-4-Kanal für Annexin (PMT 765, logarithmischer Maßstab) Dot-Plot, durch Erfassen eines Signals, bis 20.000 Zählen Perlen sind in Tor 2 erreicht. </li><li> Ermitteln Sie die positive Annexin Ereignisse LMPs in Annexin-Puffer, der CaCl 2, und dann die Ereignisse des LMPs in Annexin Puffer subtrahieren ohne CaCl 2 (Negativkontrolle). </li><li> Berechnung der absoluten Anzahl der MPs basierend auf der folgenden Gleichung: <img alt="Gleichung 1" fo:content-width="4in" src="/files/ftp_upload/52651/52651eq1.jpg" /></li></ol> 1.3) Bestimmung des MP Protein Konzentration (Bradford-Assay) <ol><li> Bereiten 5 Reihenverdünnungen eines Proteinstandard 1,25-20 ug / ml. Pipette 800 ul jeder Standard- und Probenlösung in ein sauberes Reagenzglas in zweifacher Ausfertigung. Gib 200 ul Brad Farbstoffreagens zu jedem Röhrchen. Gut mischen, dann bei Raumtemperatur für 5 min. </li><li> Messung der Extinktion bei 595 nm. Bestimmung der Proteinkonzentration von LMPs der linearen Regression der Standardkurve. </li></ol> 2. Bronchial Gewebeexplantaten und LMPs Behandlung HINWEIS: Achten Sie besonders auf die sterile Arbeitsumgebung, und aseptisch bereiten die Lösungen und mittel folgenden Experimenten verwendet. Um die vollständige Heilung Medium vorbereiten, 1 ml der Gewebeheilung Mittelergänzung mit Serum (auf Eis aufgetaut) auf 100 ml Gewebeheilung Medium und gut mischen. 2.1) Herstellung von Bronchialgewebe Explantate <ol><li> Vor dem Kultivieren zerkratzen 6 Bereiche von 1 cm 2jeweils an der Kante der Fläche von je 100 mm Gewebekulturschale mit einem Skalpell. Coat jeweils zerkratzt 100 mm Gewebekulturschale mit 2 ml der Kulturschale Beschichtungslösung und Inkubation wird die Schale in einem befeuchteten CO 2 -Inkubator O / N bei 37 ° C. Vakuum saugen Sie den Überschusslösung und füllen Sie die Schale mit 15 ml Gewebewaschmedium. </li><li> Euthanize C57BL / 6-Mäuse (5-7 Wochen alt) durch CO 2 Inhalation nach Protokollen, die von der Tierpflege Ethik-Kommission genehmigt. </li><li> Aseptisch sezieren Lungengewebe mit Skalpell, Dumont superfeinen Pinzette und chirurgische Schere. Parenchym und Blutgefäße vorsichtig entfernen. Zeigen Lungengewebe in eiskaltes Gewebewasch Medium für den Transport ins Labor, falls zutreffend. </li><li> Ferner sezieren Bronchus im Tissue Waschmedium getaucht und trenne die Bronchien mit einem Durchmesser von 1 bis 2,5 mm vom peripheren Lungengeweben. Scheibe Bronchialgewebe in ca. 5 mm dick Bronchien Ringe mit einem Skalpell. </li&gt; <li> Verwenden Sie einen Schöpfbewegung mit den sterilen gebogenen microdissecting Pinzette zu holen die bronchiale Fragmente und legen Sie sie auf die verkratzten Stellen der Gerichte. </li><li> Entfernen Sie die Gewebewaschmedium und bebrüten die Fragmente bei RT ~ 5 Minuten, damit sie an die Gerichte zu halten. </li><li> 10 ml komplette Heilung Medium zu jeder Schale und legen Sie sie in einer kontrollierten Atmosphäre modulare Inkubatorkammer. Spülen Sie die Kammer mit hoher O 2 Gasgemisch (70% O 2, 25% N 2 und 5% CO 2). Legen Sie die Kammer in einem Benchtop Orbital-Inkubator und schütteln Sie sie bei 37 ° C. Schüttle die Kammer für 24 h bei 10 Zyklen pro Minute, bis das Medium, um intermittierend über die Fragmente fließen. </li><li> Nach 24 Stunden Inkubation, beachten Sie Gewebeexplantaten unter einem Phasenkontrast-invertierten Lichtmikroskop. Wählen bronchiale Explantate mit kompletter, feines Haar Bewegung und lebendige Bronchialepithel für nachfolgende LMPs Behandlung. </li></ol>2.2) LMPs Behandlung <ol><li> Bereiten komplette Wachstumsmedium wie folgt: Tau-Wachstumsmedium ergänzt mit Serum und Fibroblasten-Inhibitor auf Eis. 1 ml des Wachstumsmediums ergänzt mit Serum und 200 ul Fibroblasteninhibitor zu 100 ml des Wachstumsmediums; gründlich mischen. Erwärmen die vollständige Wachstumsmedium bei 37 ° C für 10 min vor der Verwendung. </li><li> Verdünne isoliert LMPs in ein neues steriles Eppendorf-Röhrchen mit PBS auf eine LMPs Lager in einer Konzentration von 800 ug / ml herzustellen. </li><li> Mit 0,5 ml vollständigem Wachstumsmedium in jede Vertiefung einer 12-Well-Gewebekulturplatte. </li><li> Übertragung der gewählten bronchiale Explantate mit den gekrümmten microdissecting Pinzette aus dem vorherigen Protokoll (Abschnitt 2.1) in jede Vertiefung der Gewebekulturplatte. </li><li> Beschriften Sie die Kulturplatte angemessen auf LMPs Behandlung Brunnen und Kontrollvertiefungen zu identifizieren. In 25 ul LMPs Lager ineinander LMPs Behandlung gut (für eine Endkonzentration von 40 ug / ml) und 25 ul Kontrollfahrzeug (siehe LMP<html> s-Produktion) zu den Kontrollvertiefungen. </li><li> Fortsetzung der Inkubation in einer kontrollierten Atmosphäre modularen Inkubatorkammer bei 37 ° C unter leichtem Schütteln. </li><li> Nach 24 Stunden, waschen Explantate 3-mal mit PBS und fahren Sie mit dem nächsten Schritt (4% Paraformaldehyd [PFA] Fixierung). </li></ol> 3. Histopathologische Untersuchung 3.1) die folgenden Lösungen an, bevor Sie den nächsten Schritte <ol><li> Bereiten 1x PBS-Puffer hergestellt, indem 137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4, 1,76 mM KH 2 PO 4, pH 7,4. </li><li> Die Herstellung von 4% PFA, aufzulösen 20 g PFA in 400 ml Wasser, bei 60 ° C unter Rühren erhitzt; ein paar Tropfen von 10 M NaOH, um die Lösung zu löschen. Next hinzufügen 1x PBS-Puffer und die Lautstärke auf 500 ml und pH-Wert auf 7,4. Filter und aliquoten; bei -20 ° C. </li><li> Bereiten Sie die folgenden Austrocknung oder Rehydrierung Reagenzien; 100%, 90%, 70%, 50% Ethanol und Xylol. </li></ol>e_step &quot;&gt; 3.2) Explantation Fixierung und Gewebeschnitten Entparaffinierung <ol><li> Legen Sie jedes Explantat in einem markierten Mikrozentrifugenröhrchen mit 1,5 ml 4% PFA und Inkubation O / N bei 4 ° C. Zweimal Spülen Sie die Explantate mit 1x PBS. </li><li> Entwässern Explantate durch eine Alkoholreihe (70% Ethanol: jeder 3-mal 30 Minuten, 90% Ethanol: je 2 mal 30 min; 100% Ethanol: 3 mal 30 Minuten jeweils, dann Xylol: 3 mal 20 Minuten jede). Führen Sie alle Schritte bei Raumtemperatur in einem Abzug. </li><li> Imbed Gewebeexplantate in Paraffin bei 58 ° C in einem Ofen. Bereiten Sie 5 um dicken Gewebeschnitten unter Verwendung eines Rotationsmikrotoms. </li><li> Schweben Sie die Abschnitte in einem 56 ° C Wasserbad, und montieren Sie die Schnitte auf beschriftet histologischen Präparaten. Objektträger in manuelle Färbung Racks und trocken bei 65 ° C für 1 Stunde. Die Objektträger bei Raumtemperatur abkühlen lassen. </li><li> Tauchen Sie die Racks in 4 aufeinander folgenden Fleckenschalen mit Xylol 10 min je nach Paraffin zu entfernen. Tauchen die Racks mit einer Ethanolserie Entfernung von Xylol: 100%, dann 95%, then 80%, dann 70%, dann 50% Ethanol (5 Minuten für jeden Schritt). Spülen Sie die Ständer mit Leitungswasser für 5 Minuten, um Ethanol zu entfernen. </li></ol> 3.3) Hämatoxylin und Eosin (H &amp; E) Färbung <ol><li> Weiterhin mit den Gewebeschnitten; legen die Zahnstange in einen Färbetrog mit Mayers Hämatoxylin gefüllt für 15 min. Spülen Sie das Rack mit Leitungswasser auf Hämatoxylin für 20 Minuten zu entfernen. </li><li> Platzieren in destilliertem Wasser für 30 sec.Place in 95% Ethanol für 30 sec. Platzieren Sie in Eosin G Färbelösung Gericht für 1 min. Dehydratisieren durch 2 Änderungen von 95% Ethanol, 100% Ethanol und Xylol, jeweils 2 min. </li><li> Führen Sie eine schnelle Überprüfung unter dem Mikroskop, damit überschüssiges Eosin wird entfernt. Zeigen 2-3 Tropfen Eindeckmedium (Fisher SP15-100) auf jeder Folie, dann decken mit einem Deckglas. </li></ol> 3.4) In-situ-Cell Death Detection: TUNEL Assay <ol><li> Bevor Sie beginnen, bereiten Proteinase K Arbeitslösung: 20 ug / ml in10 mM Tris / HCl, pH 7,4. </li><li> Wiederholen Sie die Schritte 1 bis 5 von Ziffer 3.2 (Explantation Fixierung und Gewebeschnitt Entparaffinierung). Spülen Sie die Folien mit deionisiertem H 2 O </li><li> Tauchen Sie die Objektträger mit 1x PBS für 10 min. Lassen Sie das überschüssige PBS. Inkubieren Gewebeschnitten 30 min bei RT mit Proteinase K-Arbeitslösung. Rinse gleitet zweimal mit 1 × PBS. </li><li> Führen Sie die TUNEL-Assay, wie in der Bedienungsanleitung der Zelltod-Nachweis-Kit beschrieben. Montieren Sie mit Eindeckmedium und Deckglas von Hand mit Deckgläsern. </li><li> Analysieren Sie die Proben unter einem Lichtmikroskop. Verwenden Sie Image Pro 4.5, um die apoptotischen Zellen in der braunen Farbe analysieren. </li></ol>

<ol>
	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, 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-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+plasma+membrane-derived+microparticles.">Therapeutic potential of plasma membrane-derived microparticles.</a> Pharmacol Rep. 61 (1), 49-57 (2009).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways.">Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.</a> Am J Physiol Regul Integr Comp Physiol. 294 (2), 467-476 (2008).</li><li>Yang, C., Gagnon, C., Hou, X., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells.">Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.</a> Cancer Biol Ther. 10 (5), 448-456 (2010).</li><li>Angelillo-Scherrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-derived+microparticles+in+vascular+homeostasis.">Leukocyte-derived microparticles in vascular homeostasis.</a> Circ Res. 110 (2), 356-369 (2012).</li><li>Maeno, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> J Immunol. 178 (12), 8090-8096 (2007).</li><li>Qiu, Q., Xiong, W., Yang, C., Gagnon, C., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte-derived+microparticles+induce+bronchial+epithelial+cells'+pro-inflammatory+cytokine+production+and+apoptosis.">Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.</a> Mol Immunol. 55 (3-4), 220-230 (2013).</li><li>Martin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shed+membrane+particles+from+T+lymphocytes+impair+endothelial+function+and+regulate+endothelial+protein+expression.">Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.</a> Circulation. 109 (13), 1653-1659 (2004).</li><li>Shet, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+blood+contains+tissue+factor-positive+microparticles+derived+from+endothelial+cells+and+monocytes.">Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes.</a> Blood. 102 (7), 2678-2683 (2003).</li><li>Mause, S. 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Die Autoren haben nichts zu offenbaren.

Abgeordnete sind aktive Vermittler der interzellulären Übersprechen und deren Studie ist in vielen Bereichen der Wissenschaft verspricht. 11 Diese Studie präsentiert ein detailliertes Protokoll für die in-vitro-Groß Generation LMPs von einem apoptotischen T-Zelllinie abgeleitet. Diese Abgeordneten zum Ausdruck bringen ein großes Repertoire an Lymphozyten-Molekülen und sind biologisch in der Regulation von Zell- und Gewebe Homöostase beteiligt ist. Jedoch kann LMPs aus verschiedenen Quellen ab...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">LMPs production and characterization</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CEM T cells&#160;</td>
			<td style="width:231px;">ATCC&#160;</td>
			<td style="width:124px;">CCL-119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">X-VIVO 15 medium&#160;</td>
			<td style="width:231px;">Cambrex, Walkersville</td>
			<td>04-744Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T75</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1813.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T175</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1812.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Actinomycin&#160;D&#160;</td>
			<td style="width:231px;">Sigma Chemical Co.</td>
			<td style="width:124px;">A9415-2mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PBS</td>
			<td style="width:231px;">Lifetechnologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#181;m filter</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1826.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Annexin-VCy5</td>
			<td style="width:231px;">BD Pharmagen&#160;</td>
			<td>559933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS flow solution</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">342003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fluorescent microbeads (1 um)</td>
			<td style="width:231px;">Molecular Probes&#160;</td>
			<td style="width:124px;">T8880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysterene counting beads (7 um)</td>
			<td style="width:231px;">Bangs laboratories</td>
			<td style="width:124px;">PS06N/6994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene FACS tubes</td>
			<td>Falcon</td>
			<td style="width:124px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1 ml pipet</td>
			<td style="width:231px;">Fisher</td>
			<td style="width:124px;">13-678-11B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">5 ml pipet</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:124px;">357543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">25 ml pipet</td>
			<td style="width:231px;">Ultident</td>
			<td style="width:124px;">DL-357551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1,5 ml conical polypropylene micro tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">72.690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.554.205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.547.205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">50 ml high speed polypropylene copolymer tube</td>
			<td style="width:231px;">Nalgene</td>
			<td style="width:124px;">3119-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">250 ml high speed polypropylene bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">356011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay (Bradford assay)</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay standard II</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 16x100</td>
			<td>VWR</td>
			<td>47729-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 12x75</td>
			<td>Ultident</td>
			<td>170-14100005B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator&#160;</td>
			<td style="width:231px;">Mandel</td>
			<td style="width:124px;">Heracell 150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Low speed centrifuge</td>
			<td style="width:231px;">IEC</td>
			<td style="width:124px;">Centra8R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed centrifuge</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Avanti J8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 250ml bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JLA16.250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 50ml tube</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JA30.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fow cytometry&#160;</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">FACS Calibur</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Spectrophotometer</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Series 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bronchial tissue explants and sections&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice (5-7 weeks old)&#160;&#160;</td>
			<td style="width:231px;">Charles River Laboratories, Inc.&#160;</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Airway PrimaCell&#8482; System:</td>
			<td style="width:231px;">CHI Scientific, Inc.</td>
			<td style="width:124px;">2-82001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Rib-Back Carbon Steel Scalpel Blades</td>
			<td style="width:231px;">Becton Dickinson AcuteCare</td>
			<td style="width:124px;">371310</td>
			<td>#10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">10003-12</td>
			<td>#7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phase-contrast inverted microscope</td>
			<td style="width:231px;">Olympus Optical CO., LTD.&#160;&#160;&#160;</td>
			<td style="width:124px;">CK2</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">high O2 gas mixture&#160;</td>
			<td>VitalAire Canada Inc.</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">modular incubator chamber</td>
			<td style="width:231px;">Billups-Rothenberg Inc.</td>
			<td style="width:124px;">MIC-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ 4000 incubated orbital shaker</td>
			<td style="width:231px;">Barnstead Lab-Line,&#160;</td>
			<td style="width:124px;">SHKA4000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well tissue culture plate</td>
			<td style="width:231px;">Becton Dickinson and Company</td>
			<td style="width:124px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plastic tissue culture dishes (100 mm)</td>
			<td style="width:231px;">Sarstedt, Inc.</td>
			<td style="width:124px;">83.1802</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Surgical scissors</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">14060-09</td>
			<td style="width:171px;">Straight, sharp, 9cm longth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Half-curved Graefe forceps</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">11052-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">humidified CO2 incubator</td>
			<td style="width:231px;">Mandel Scientific Company Inc.&#160;</td>
			<td style="width:124px;">SVH-51023421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Histopathological examination&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formalin formaldehyde</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">T555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethyl alcohol</td>
			<td style="width:231px;">Merck KGaA, Darmstadt</td>
			<td style="width:124px;">EX0278-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;glutaraldehyde&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">G6403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cacodylate</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">31533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope slides</td>
			<td style="width:231px;">VWR Scientific Inc.&#160;</td>
			<td style="width:124px;">48300-025</td>
			<td>25x75mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">X5-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayer&#39;s hematoxylin</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">MHS16</td>
			<td>Funnel with filter paper&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl&#160;</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.&#160;&#160;</td>
			<td style="width:124px;">A144s-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eosin&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT110116</td>
			<td>Funnel with filter paper&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permount&#8482; Mounting Medium</td>
			<td style="width:231px;">Thermo Fisher Scientific Inc.&#160;</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass coverslip</td>
			<td style="width:231px;">surgipath medical industries, Inc.</td>
			<td style="width:124px;">84503</td>
			<td>24&#215;24&#160;#1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TUNEL detection kit</td>
			<td style="width:231px;">In Situ Cell Death Detection, POD</td>
			<td style="width:124px;">11 684 817 910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">oven</td>
			<td style="width:231px;">Despatch Industries Inc.</td>
			<td style="width:124px;">LEB-1-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">rotary Microtome</td>
			<td style="width:231px;">Leica Microsystems Inc.</td>
			<td style="width:124px;">RM2145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">filter paper</td>
			<td style="width:231px;">Whatman International Ltd.</td>
			<td style="width:124px;">1003150</td>
			<td>#3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:231px;">Nikon Imaging Japan Inc.</td>
			<td style="width:124px;">E800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">staining dish complete</td>
			<td style="width:231px;">Wheaton Industries, Inc.</td>
			<td>900200</td>
			<td>including dish, rack, cover</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1.5 ml eppendorf tube</td>
			<td style="width:231px;">Sarstedt Inc.&#160;</td>
			<td style="width:124px;">72.69</td>
			<td>39x10mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital and Reciprocating Water Bath</td>
			<td style="width:231px;">ExpotechUSA</td>
			<td style="width:124px;">ORS200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate buffered saline&#160;&#160;</td>
			<td style="width:231px;">GIBCO</td>
			<td style="width:124px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fume hood</td>
			<td>Nicram RD Service</td>
			<td>3707E</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

Das Interesse an der biologischen Rolle von Zellmembranen abgeleiteten Vesikeln in Zell-Zell-Kommunikation in den letzten Jahren erhöht. Mikroteilchen (MPs) sind eine solche Art der Bläschen, deren Durchmesser von 0,1 um bis 1 um und typischerweise von der Plasmamembran von eukaryotischen Zellen, die Aktivierung oder die Apoptose zu vergießen. Hier beschreiben wir die Erzeugung von T-Lymphozyten abgeleiteten Mikroteilchen (LMPs) von apoptotischen CEM T-Zellen mit Actinomycin D. LMPs werden durch einen mehrstufigen Differenz Zentrifugationsverfahren isoliert und mittels Durchflusszytometrie stimuliert. Dieses Protokoll stellt auch eine in situ Zelltod Nachweismethode für den Nachweis der proapoptotischen Wirkung LMPs auf bronchialen Epithelzellen aus Maus primären Atem bronchiale Gewebeexplantaten abgeleitet. Hierin beschriebene Verfahren eine reproduzierbare Verfahren zur Isolierung reichliche Mengen von LMPs apoptotische Lymphozyten in vitro. LMPs abgeleitetin dieser Weise verwendet werden, um die Eigenschaften der verschiedenen Krankheitsmodellen zu bewerten und für pharmakologische und toxikologische Prüfung. Da das Atemwegsepithel bietet eine Schutz physische und funktionelle Barriere zwischen der äußeren Umgebung und der darunter liegenden Gewebe, stellt die Verwendung von Bronchialgewebe Explantate nicht immortalisierte Epithelzellen Linien ein effektives Modell für Untersuchungen Atemwegstrakt Gewebe erfordern.

LMPs wurden mit Annexin V-Färbung 10 durch fluoreszenzaktivierte Zellsortierung (FACS) Analyse und gated mit 1 &amp; mgr; m-Perlen, in dem 97% der Abgeordneten (≤1 um) waren Annexin-V-positiven Cy5 gekennzeichnet (Abbildung 1A und 1B). Typischerweise wurden etwa 2,5 mg LMPs nach diesem Protokoll erhalten. Bronchialgewebe Explantaten aus C57BL / 6-Mäuse wurden zum Träger und LMPs Behandlung unterworfen. Histopathologische Analyse Bronchialsegmente zeigte die Wirkung von LMPs auf die struk...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Zellmembran-Schuppen-Mikropartikel (MP) aktiv biologischen Vesikel, die isoliert werden können und deren pathophysiologische Effekte in verschiedenen Modellen untersucht. Hier beschreiben wir ein Verfahren zum Erzeugen MPs von T-Lymphozyten (LMPs) abgeleitet und zur Demonstration ihrer proapoptotischen Effekt auf Epithelzellen der Luftwege.

Generierung von lymphatische Mikropartikel und Nachweis ihrer pro-apoptotische Wirkung auf die Epithelzellen der Atemwege

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

generation-lymphocytic-microparticles-detection-their-proapoptotic

Research

JoVE Journal

Immunology and Infection

8.9K Aufrufe.  University of Montréal.  Zellmembran-Schuppen-Mikropartikel (MP) aktiv biologischen Vesikel, die isoliert werden können und deren pathophysiologische Effekte in verschiedenen Modellen untersucht. Hier beschreiben wir ein Verfahren zum Erzeugen MPs von T-Lymphozyten (LMPs) abgeleitet und zur Demonstration ihrer proapoptotischen Effekt auf Epithelzellen der Luftwege. 

Serie: Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-Kultur Modelle Pseudomonas aeruginosa Biofilme auf lebenden menschlichen Zellen gezüchtet Airway

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Forschung

Immunologie und Infektion

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Die Anwendung eines Maus Ligierte Peyer-Patch-Darmschlinge Assay zu bakterielle Aufnahme von M-Zellen auswerten

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Aufbau einer Liquid-bedeckten Kultur Polarized Menschliche Airway Epithelial Calu-3-Zellen, um Host-Zell-Antwort, um Atemwegspathogene Studie<em&gt; In-vitro-</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Methoden für die quantitative Bestimmung von Antikörper-induzierte Komplementaktivierung auf Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Groß Reinigung von Schwein oder Rind Photorezeptoraußensegmente für Phagozytose Assays auf Netzhautpigmentepithelzellen

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Die phänotypische und funktionale Analyse von aktivierten regulatorischen T-Zellen isoliert von chronischer lymphatischer Choriomeningitisvirus-infizierte Mäuse

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Nicht-invasive Probenahme von Schleimhaut-Flüssigkeit für die Quantifizierung von In Vivo oberen Atemwegen immun-Mediator-Level

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Isolierung und Charakterisierung von Neutrophilen abgeleitet Mikropartikel für funktionelle Studien

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...