Die Autoren möchten sich Anne Daron und Pierre Louis Loyher für redaktionelle Unterstützung danken, die Plateforme Imagerie Pitié-Salpêtrière (PICPS) für die Unterstützung bei der Zwei-Photonen-Mikroskop und das Tier Facility &quot;NAC&quot; und Camille Baudesson für Mäuse züchten Unterstützung. Die Forschung, die zu diesen Ergebnissen geführt Mittel aus dem Siebten Rahmenprogramm der Europäischen Gemeinschaft (FP7 / 2007-2013) unter Finanzhilfevereinbarung Nr 304.810 - RAIDs und Nr 241.440-EndoStem von Inserm, der Université Pierre et Marie Curie &quot;Emergence &quot;, von la&quot; Ligue contre le cancer &quot;, von&quot; Association pour la Recherche sur le Cancer &quot;und von&quot; Agence Nationale de la Recherche &quot;Programm Emergence 2012 (ANR-EMMA-050). PH wurde von la &quot;Ligue contre le cancer&quot; unterstützt.

HINWEIS: Alle Experiment Protokolle wurden von der Französisch Tierversuche und Ethik-Kommission genehmigt und vom &quot;Service Protection et Santé Animales, Environnement&quot; mit der Nummer A-75-2065 validiert. Probengrößen werden so gewählt, um die Reproduzierbarkeit der Experimente zu gewährleisten und nach dem 3R tierischen Ethik Regulierung. 1. Herstellung der Mäuse <ol><li> Anästhesie <ol><li> Für kurze Zeit der Bildgebung (weniger als 1 Stunde), betäuben die Maus mit einer intraperitonealen Injektion von 200 ul einer Kochsalzlösung mit Ketamin (100 mg / kg) und Xylazin (10 mg / kg). </li><li> Alternativ kann für längere Zeitabbildung (bis zu 4 h), anaesthetize der Maus bei einer Inhalation von Isofluran 2,5% in einer 70/30 Mischung von O 2 / N 2 O, verdampft durch eine angepasste Maske. </li><li> HINWEIS: Auf diese Weise sorgt für stabilere Bewusstlosigkeit und vermeidet Serien intraperitoneale Injektion von Drogen. </li&gt; <li> Sobald die Maus betäubt, überprüfen Bewusstlosigkeit durch Stimulation der Fußsohle mit Zangen. </li></ol></li><li> Gefäßfärbung / zusätzliche Färbung <ol><li> Für vaskuläre Färbung: intravenös injizieren in die Schwanzvene wurden 200 ul Rhodamin-Dextran (2 MDa, 10 mg / ml in Kochsalzpuffer). </li><li> Für Neutrophilen-Färbung: injizieren 2 ug Ly6G-PE (Klon 1A8) in 100 ul Kochsalzlösung, intravenös in die Schwanzvene. </li></ol></li><li> Immobilisierung <ol><li> Zur Immobilisierung, verwenden Sie eine maßgeschneiderte stereotaktischen Halterung an das Mikroskop und mit dem Narkosegas Inhalator angepasst. HINWEIS: Die stereotaktische Halter ist ein Metallträger mit zwei Metallplatte Klammern, von denen einer fest und der andere Wechsel und anpassungsfähig, um den Kopf des Tieres fest. </li><li> Installieren Sie den Kopf der Maus zwischen den Halteplatten isoliert von Atembewegungen zu gewährleisten. Ziehen Sie die Ohren zwischen Halteplatten und den Kopf auf die sk streckenin. Prüfen Sie für die Stabilität und die Atmung Wohlbefinden der Tiere. </li></ol></li><li> Entfernen Sie die Kopfhaut <ol><li> Vorsichtig mit 70% igem Ethanol, um das Haar von der Kopfhaut mit einem sterilen Applikators benetzen, Vermeidung von Kontakt mit den Augen. </li><li> Schneiden Sie die Haut mit einer sterilen Schere und Zange, die Kopfhaut von der Rückwärts des Scheitelbein vollständig zu entfernen, um das Stirnbein. Fernhalten von bis zu 3 mm von Augen und Ohren, um umfangreiche Blutungen zu verhindern. Entfernen Sie die Abdeckung des Schädels (Knochenhaut) lockerem Bindegewebe. Waschen Sie die restlichen Haare mit warmem PBS getränkten Papiertuch. </li></ol></li><li> Immersion-System einrichten <ol><li> Fügen Sie einen Gummiring von 18 mm Durchmesser mit chirurgischer Kleber direkt auf den Schädel mit einer kleinen Nadel zu Verteilung zu steuern. Kleben den gesamten Umfang des Gummirings, um das Auslaufen zu verhindern (30 &amp; mgr; l ausreichend sein). </li><li> Reinigen Sie den Schädel unter dem Ring ein letztes Mal mit PBS-getränkten Papiertuch und fügen Sie dann 37 ° C PBS für ein vollständiges Eintauchen derSchädel. Fügen Sie einen Tropfen 0,9% NaCl-Lösung oder bestimmte Augensalbe auf jedem Auge der Maus an Augentrockenheit zu vermeiden. Ziehen Sie den stereotaktischen Halterung im Rahmen des Ziels. </li><li> Etwa positionieren Sie den Bereich mit dem Mikroskop Okular durch direkte Übertragung. </li></ol></li></ol> 2. Zwei-Photonen-Imaging Acquisition <ol><li> Verwenden einer Multiphotonen-Mikroskop in Verbindung mit einem Ti: Saphir-Kristall-Laser, der Impulse von 140fs NIR-Licht enthält, selektiv abstimmbar von 680 bis 1.080 nm und einen akusto-optischen Modulator für die Laserleistungssteuerung. Verwenden Sie eine Konfiguration mit 3 externen nichtdescannten Detektoren (NDD) mit einer Kombination von zwei dichroitischen Spiegel (565 nm und 690 nm), 565/610 und 500/550 Bandpassfilter (die gleichzeitige Aufnahme von 3 Fluoreszenzkanäle zu ermöglichen), und einen 485 Kurzpassfilter, mit einem Plan Achromat × 20 (NA = 1) Wasserimmersionsobjektiv. </li><li> Stellen Sie den Heizraum, gute homeothermy der ANAES ermöglichenthetized Maus., HINWEIS: Temperatur vielleicht 1 Stunde eingestellt werden, bevor die Abbildung Sitzung für eine bessere Stabilität. In unserem Fall 32 ° C wurde gewählt, um eine optimale Körpertemperatur des anästhesierten Maus (36-37 ° C) erhalten und ist für bis zu 4 h gehalten. Diese Temperatur kann mit dem System verwendet (digital Sonde kann verwendet werden, um diesen Punkt zu überprüfen) angepasst werden. Darüber hinaus ist das verwendete Heizkammer dunkel / schwarz und deckt einen Teil des Systems (Ziele und motorisierten Platte) fremdlicht Kontamination zu vermeiden. </li><li> Schalten Sie das System <ol><li> Einschalten des Ti: Saphir-Laser, dann das gesamte Mikroskopsystem. Starten Sie die Erfassungssoftware. Stellen Sie den Laser-Wellenlängenbereich um 870 nm. Wählen Sie die entsprechende NDD für die Aufnahme. HINWEIS: In unserem Fall sind Erzeugung der zweiten Harmonischen (SHG) 19 und cyan fluoreszierende Protein (ECFP) durch die NDD nach 485 Kurzpassfilter detektiert. ECFP wird auch durch die NDD detektiert, nachdem die 500/550 Bandpaßfilter und Rhodamin-Dextran wird durch t detektierter NDD nach dem 565/610 Bandpassfilter. </li></ol></li><li> Passen Akquisition Einstellungen <ol><li> Verwenden Sie die &quot;Live-Akquisition&quot; Imaging-Befehl der Software und mit dem Mikroskop Wege, stellen Sie den Fokus auf eine Region mit ECFP und Rhodamin-Signal. Eingestellt, die Laserleistung auf ein Minimum, um das beste Signal-zu-Rausch-Verhältnis bei minimalen Photoschäden zu erhalten. HINWEIS: Die Leistungseinstellung variiert je nach der Tiefe der Bereich von Interesse. Typischerweise werden die AOM-Einstellungen werden rund 20%, die eine Laserleistung über das Ziel um 15 mW repräsentiert gesetzt. Es ist besser, Gewinn Macht auf den PMTs erhöhen für jeden NDD an Photobleaching und Foto-Schaden zu reduzieren. Typische Erfassungseinstellungen einzigen Abtastung mit einem Pixelverweilzeit von 1,58 &amp; mgr; s und einer Auflösung von 512 x 512 Pixeln mit einer 1,5 Vergrößerung. </li></ol></li><li> Wählen Sie die Regionen von Interesse <ol><li> Sobald die Aufnahme Parameter eingestellt sind, verwenden Sie erneut die &quot;live&quot; Erwerb Befehl,und Erhebung des Gewebes, um eine gewünschte (x, y, z) der Wert in Echtzeit überwacht werden soll. HINWEIS: In der Regel haben wir uns für ein Feld sowohl vaskuläre und parenchymale Bereichen. </li><li> Registrieren Sie das Feld mit der &quot;Position&quot; Softwarebefehl. ANMERKUNG: Dieser Befehl speichert x, y, z-Koordinaten der verschiedenen entfernten Bereichen. </li><li> Wählen und registrieren zusätzliche Felder von Interesse (bis zu 3) nach dem gleichen Verfahren. </li><li> Festlegen eines 3D-Z-Stapel mit dem &quot;Z-Stapel&quot; Softwarebefehl auf der ersten gespeicherten Position entsprechend der gewünschten Lautstärke mit gewählten Vergrößerung. Merken tieferen Position zuerst, und dann die höchste Position. Normalisieren Laserleistung mit der Tiefe. HINWEIS: Bei mehreren gleichzeitigen Feldabbildung, so erwerben wir 5 Scheiben von 3 &amp; mgr; z-Schritte für jedes Feld getrennt werden, um ein Volumen von 12 &amp; mgr; m Dicke zu erwerben. Diese Einstellungen werden auf die Art der Zelle untersucht angepasst werden. </li></ol></li><li> Starten Erwerb <ol><li> Wählen Sie das220, Reihe &quot;Software-Befehl und wählen Sie die Dauer des Zeitintervalls und die Anzahl der Zyklen der Zeit. Beginnen Sie Zeitraffer-Bildgebung nach dem Zeitablauf und Dauer, indem Sie die &quot;Start-Experiment&quot; Software-Befehl erforderlich. HINWEIS: In unserem Fall haben wir ein Volumen jeweils 30 s bei 60 Hz entspricht 30 min Echtzeit zu erwerben. Kleinere Zeitablauf durchgeführt, um schnelle Zellvoll in das Blutgefäß zu verfolgen. In diesem Fall dünner z-Stapels und nicht mehr als 2 verschiedenen Felder können gleichzeitig aufgezeichnet werden. </li><li> Eine regelmäßige Überwachung der Einrichtung. <ol><li> Immer überwachen das Tier, um sicherzustellen, sie bewusstlos ist. HINWEIS: Schütteln der Whisker oder Beine des Tieres sind Indikatoren für die Wiederbelebung. Im Falle des Tieres Atmung ruckartig, kann die Menge von Isofluran leicht reduziert werden. Starke Gewebedrift während Akquisition ist ein Indikator für Maus Revival. </li><li> Stellen Sie sicher, das Ziel richtig eingetaucht. Erneuern 37ºC PBS between zwei Akquisitionen (30 min). HINWEIS: Progressive Verschwinden des Signals während der Erfassung ist Indikator für die PBS Leckage. </li></ol></li></ol></li><li> Ende der Prozedur <ol><li> Wenn die benötigten Videos aufgezeichnet werden, das Tier einschläfern durch Genickbruch schnell vor Reanimation. HINWEIS: Wundheilung führt zur Bildung einer Kruste in 24 bis 48 h, was die Möglichkeit zur Längs- Bildgebung auszuführen begrenzt. </li></ol></li></ol> 3. Datenanalyse <ol><li> Driftkorrektur <ol><li> Verwenden Imaris Bitplane Software für 3D-automatische Verfolgung. </li><li> Öffnen Sie die 3D-Bildfolge auf Imaris. Wählen Sie &quot;Spot&quot; Befehl ein, und erzeugen eine manuelle Spur (Mausklick + Shift) für alle Zeitpunkte für mindestens 4 verschiedene Ankerpunkte, möglichst homogen im Feld verteilt. </li><li> Übernehmen Sie die Driftkorrektur mit dem Befehl &quot;richtigen Drift&quot; im Register &quot;Bearbeitungsspur&quot;. </li><li> Prüfen Sie, ob ter Qualität der Korrektur in x, y und z, um vor allem in Artefakt Schwanken zu vermeiden. HINWEIS: Wenn die Korrektur nicht zufrieden stellend, versuchen andere Ankerpunkte oder ausschließen das Video von der Analyse. </li></ol></li><li> Zellverfolgung <ol><li> Verwenden Sie &quot;add new spots&quot; Befehl und wählen Sie &quot;Spur-Spots im Laufe der Zeit&quot; Algorithmus-Einstellung. Zum nächsten Schritt. </li><li> Bewerben automatischen Zelltrackingverfahren entweder auf der gesamten interessierenden Kanal mit &quot;Quellkanal&quot; -Befehl. Gruppenobjekte (dh Zellen) Durchmesser bis 10 um. Zum nächsten Schritt. HINWEIS: Die Wahl wird auf die Unterscheidung zwischen Objekten und Spezifität des Messsignals abhängen. Kurz gesagt, ist die automatische Verfolgung im ganzen Kanal nur möglich, wenn erfasste Signal spezifisch für das Objekt von Interesse ist. Da sowohl SHG und ECFP werden von der NDD nach 485 Kurzpassfilter erkannt wird, wird Tracking präziser und effizienter mit dem ECFP Signals durch die NDD nach der 5 aufgenommen sein00/550 Bandpassfilter. Gruppierten oder dichtgepackten Objekte manuelle Auswahl der ähnlich wie in Schritt 3.1.2 einzelne Objekte erfordern. </li><li> Wählen Sie &quot;Qualität&quot; Filtertyp können Sie den Schwellwert auf Verfolgung von unspezifischen Objekte ausschließen. Zum nächsten Schritt. </li><li> Wählen Sie &quot;Brownschen Algorithmus Motion&quot; mit genauen Parameter &quot;Max Abstand&quot; und &quot;Gap Größe&quot; zwischen zwei Punkten. Bestätigen Track Generation. </li><li> Sobald Spuren werden automatisch berechnet, &quot;Spurdauer&quot; Filtertyp auswählen, stellen Sie den Schwellenwert auf Spuren zu beseitigen kürzer als 120 Sekunden (die 4 Zeitpunkte). </li><li> Obligatorisch: Nach dem automatischen Verfolgung, prüfen Sie die Qualität der Tracks. <ol><li> Rollen Sie die Zeitleiste, um zu überprüfen, dass die Objekte folgen Bahnverläufe. Wenn nicht, verwenden Sie die Optionen der Spur Punktkorrektur in der Registerkarte &quot;Bearbeiten&quot;: Löschen Sie falsche oder unsachgemäße Spuren mit &quot;Löschen&quot; -Befehl (löschen jede Spur point einzeln mit dem Befehl &quot;Trennen&quot;). Wenn Track-Pfad unterbrochen ist, fügen fehlenden Zeitpunkten (Mausklick + Shift), wählen Sie alle Punkte auf einer bestimmten Spur, und verwenden Sie den Befehl &quot;Verbinden&quot; auf der Registerkarte &quot;Track bearbeiten&quot;. </li></ol></li></ol></li><li> Quantifizierung Parameter <ol><li> Wählen Sie die Registerkarte &quot;Statistik&quot; im Befehl &quot;Einstellungen&quot;, wählen Sie die dynamischen Parameter von Interesse in der Liste. Wählen Sie &quot;Exportieren Sie alle Statistiken in Datei&quot; Befehl und speichern Sie die Excel-Datei erzeugt. HINWEIS: Mehrere dynamische Parameter werden direkt aus der Software erhalten, wie Bahnlängen, momentane Geschwindigkeit, mittlere Geschwindigkeit und verfolgen Geradheit. Motilität Koeffizienten 6, 20 ermittelt werden kann, ist jedoch nicht direkt durch die Software zur Verfügung gestellt. </li><li> Für jede Zelle berechnet den Mittelwert der Verschiebungen für jeden Zeitraum (&amp; dgr; t) (bei einem 30 Sek Zeitrafferfilm, wird &amp; dgr; t 30, 60, 90 Sekunden, usw. sein).Zur Erstellung der Mittelwert der quadratischen Verschiebung als eine Funktion des Zeitintervalls. Erhalten Motilität Koeffizienten durch Berechnen der Steigung des linearen Teils der Kurve. </li></ol></li></ol>

<ol>
	<li>Ovchinnikov, D. A., 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=Expression+of+Gal4-dependent+transgenes+in+cells+of+the+mononuclear+phagocyte+system+labeled+with+enhanced+cyan+fluorescent+protein+using+Csf1r-Gal4VP16/UAS-ECFP+double-transgenic+mice.">Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice.</a> J Leukoc Biol. 83, 430-433 (2008).</li><li>Parihar, A., Eubank, T. D., Doseff, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+macrophages+regulate+immunity+through+dynamic+networks+of+survival+and+cell+death.">Monocytes and macrophages regulate immunity through dynamic networks of survival and cell death.</a> J Innate Immun. 2, 204-215 (2010).</li><li>Geissmann, F., Jung, S., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+monocytes+consist+of+two+principal+subsets+with+distinct+migratory+properties.">Blood monocytes consist of two principal subsets with distinct migratory properties.</a> Immunity. 19, 71-82 (2003).</li><li>Robbins, C. S., Swirski, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+roles+of+monocyte+subsets+in+steady+state+and+inflammation.">The multiple roles of monocyte subsets in steady state and inflammation.</a> Cell Mol Life Sci. 67, 2685-2693 (2010).</li><li>Shi, 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=Bone+marrow+mesenchymal+stem+and+progenitor+cells+induce+monocyte+emigration+in+response+to+circulating+toll-like+receptor+ligands.">Bone marrow mesenchymal stem and progenitor cells induce monocyte emigration in response to circulating toll-like receptor ligands.</a> Immunity. 34, 590-601 (2011).</li><li>Jacquelin, 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=CX3CR1+reduces+Ly6Chigh-monocyte+motility+within+and+release+from+the+bone+marrow+after+chemotherapy+in+mice.">CX3CR1 reduces Ly6Chigh-monocyte motility within and release from the bone marrow after chemotherapy in mice.</a> Blood. 122, 674-683 (2013).</li><li>Swirski, F. K., 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=Identification+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325, 612-616 (2009).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, 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=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat Rev Immunol. 6, 497-507 (2006).</li><li>Charo, I. F., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+chemokines+and+chemokine+receptors+in+inflammation.">The many roles of chemokines and chemokine receptors in inflammation.</a> N Engl J Med. 354, 610-621 (2006).</li><li>Milo, I., 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=Dynamic+imaging+reveals+promiscuous+crosspresentation+of+blood-borne+antigens+to+naive+CD8++T+cells+in+the+bone+marrow.">Dynamic imaging reveals promiscuous crosspresentation of blood-borne antigens to naive CD8+ T cells in the bone marrow.</a> 122, 193-208 (2013).</li><li>Cavanagh, L. L., 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=Activation+of+bone+marrow-resident+memory+T+cells+by+circulating,+antigen-bearing+dendritic+cells.">Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells.</a> Nat Immunol. 6, 1029-1037 (2005).</li><li>Mazo, I. B., 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=Bone+marrow+is+a+major+reservoir+and+site+of+recruitment+for+central+memory+CD8++T+cells.">Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.</a> Immunity. 22, 259-270 (2005).</li><li>Travlos, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+structure,+function,+and+histology+of+the+bone+marrow.">Normal structure, function, and histology of the bone marrow.</a> Toxicol Pathol. 34, 548-565 (2006).</li><li>Mazo, I. B., 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=Hematopoietic+progenitor+cell+rolling+in+bone+marrow+microvessels:+parallel+contributions+by+endothelial+selectins+and+vascular+cell+adhesion+molecule+1.">Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.</a> J Exp Med. 188, 465-474 (1998).</li><li>Rashidi, N. M., 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=In+vivo+time-lapse+imaging+of+mouse+bone+marrow+reveals+differential+niche+engagement+by+quiescent+and+naturally+activated+hematopoietic+stem+cells.">In vivo time-lapse imaging of mouse bone marrow reveals differential niche engagement by quiescent and naturally activated hematopoietic stem cells.</a> Blood. , (2014).</li><li>Sipkins, D. A., 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=In+vivo+imaging+of+specialized+bone+marrow+endothelial+microdomains+for+tumour+engraftment.">In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.</a> Nature. 435, 969-973 (2005).</li><li>Cariappa, A., 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=Perisinusoidal+B+cells+in+the+bone+marrow+participate+in+T-independent+responses+to+blood-borne+microbes.">Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes.</a> Immunity. 23, 397-407 (2005).</li><li>Junt, 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence.">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proc Natl Acad Sci U S A. 99, 11014-11019 (2002).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annu Rev Immunol. 26, 585-626 (2008).</li><li>Yost, C. 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=Impaired+neutrophil+extracellular+trap+(NET)+formation:+a+novel+innate+immune+deficiency+of+human+neonates.">Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.</a> Blood. 113, 6419-6427 (2009).</li><li>Devi, 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=Multiphoton+imaging+reveals+a+new+leukocyte+recruitment+paradigm+in+the+glomerulus.">Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.</a> Nat Med. 19, 107-112 (2013).</li></ol>

The authors have nothing to disclose.

Die kritischen Punkte der in vivo Bildgebung Methodik, um die Stabilität der Ausrichtung, um die Dauer der Bildgebung zu maximieren und das Risiko einer bakteriellen Kontamination und Entzündungen, die die Dynamik der entzündlichen Zellen auswirken können, zu minimieren gewährleisten. Imaging Schädelknochenmark folgt diese Ziele auf die Operation durchgeführt, um Zugang zu dem Knochenmark zu erhalten, ist minimal. Die Verwendung von sterilem Material und Antiseptika ist wichtig, um das Risiko von ...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

<table border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:237px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ketamin</td>
			<td style="width:171px;">Merial</td>
			<td style="width:119px;"></td>
			<td>100mg/mL, anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Xylazin</td>
			<td style="width:171px;">Bayer HealthCare</td>
			<td style="width:119px;"></td>
			<td>10mg/mL, anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Isofluran</td>
			<td style="width:171px;">Baxter</td>
			<td style="width:119px;"></td>
			<td>2.5%, anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">O2/NO2</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>70/30 mixture, anesthetic</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:297px;">Rhodamin-Dextran</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>2MDa, 10mg/mL, Vascular staining</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:297px;">Ly6G-PE</td>
			<td style="width:171px;">Becton-Dickinson</td>
			<td style="width:119px;"></td>
			<td>clone 1A8, neutrophils staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Stereotactic holder</td>
			<td style="width:171px;">Home made</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ethanol 70%</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sterile scissors and nippers</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Rubber ring</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>18mm diameter, surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glubran 2</td>
			<td style="width:171px;">Queryo Medical</td>
			<td style="width:119px;"></td>
			<td>Surgical Glue, rubber ring fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Small gauge needles</td>
			<td style="width:171px;">Terumo</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM 710 NLO multiphoton microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti:Sapphire crystal laser&#160;</td>
			<td>Coherent Chameleon Ultra</td>
			<td style="width:119px;"></td>
			<td>140fs pulses of NIR light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zen 2010</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Acquistion Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris Bitplane&#160;</td>
			<td style="width:171px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td>Analysis Software, 3D automatic tracking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PBS 1X</td>
			<td style="width:171px;">D. Dutscher</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Thermostated chamber</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>intravital imaging</td>
		</tr>
	</tbody>
</table>

tracking mouse bone marrow monocytes in vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Mäuseschädelstruktur bietet eine gute Gelegenheit, die Knochenmark Physiologie von Intravital Imaging studieren. Der Knochen dünn um den vorderen Scheitelbereich, ist es möglich, Zugang zur Mark Nischen ohne Abrieb des Knochens zu erhalten. 1 stellt eine breite 2D Bereich des Schädels eines MacBlue transgenen Maus. Die Knochenmatrix wird hauptsächlich aus Kollagen I leicht durch SHG 19 feststellbar. Injektion von Rhodamin-Dextran färbt die Gefäßnetz des Knochenmarks und ermöglicht d...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Tracking-Maus-Knochenmark-Monozyten<em&gt; In-vivo-</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.4K Aufrufe.  Sorbonne Universités, UPMC Univ Paris 06, CR7. Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism. 

Serie: Tracking Mouse Bone Marrow Monocytes In Vivo

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Erzeugung und Kennzeichnung von Murine Knochenmark gewonnenen dendritischen Zellen mit Qdot Nanokristalle für Tracking-Studien

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Forschung

Immunologie und Infektion

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziert Functional Roles of Gene Expression von Immun-und Nicht-Immunzellen in Mouse Colitis von Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolierung, Reinigung und Kennzeichnung von Knochenmark der Maus Neutrophile für funktionelle Studien und Experimente adoptiven Transfer

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Knochenmark-abgeleiteten Makrophagen-Produktion

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Murine Hind Gliedmaßen langen Knochen Dissection und Knochenmark-Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Femur Fensterkammer Modell für<em&gt; In Vivo</em&gt; Zelltracking im Knochenmark der Maus

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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

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

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

Fluoreszenz-aktivierte Zellsortierung zur Reinigung von Plasmazytoide dendritischer Zellen aus dem Knochenmark der Maus

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

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identifizierung und Isolierung von Oligopotent und Lineage-committed myeloischen Vorläuferzellen aus Maus Knochenmark

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Eine In Vivo Mausmodell Maßnahme naive CD4-T-Zell-Aktivierung, Proliferation und Th1 Differenzierung induziert durch Knochenmark-abgeleitete Dendritische Zellen

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...