This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

1. Neutrophilen-Isolierung und Co-Kultur mit primären leukämischen Zellen HINWEIS: Die Verfahren wurden im Rahmen der Genehmigung von Lyon Krankenhaus Ethikkommission bei allen Patienten durchgeführt, informierte Zustimmung zu unterzeichnen. <ol><li> Isolierung von primären leukämischen Zellen und Neutrophile <ol><li> Sammeln Sie Rohre aus dem peripheren Blut auf EDTA (1,8 mg EDTA pro Milliliter Blut) von Patienten mit chronischer lymphatischer Leukämie diagnostiziert (CLL). </li><li> Ist jeweils mit 15 ml Blut in einen sterilen 50 ml-Röhrchen und verdünnt mit 15 ml RPMI (Verdünnung 1: 1), dann vorsichtig und langsam 15 ml Dichtegradient-Lösung auf den Boden des Rohres, ohne die Phasen zu vermischen. Sicherzustellen, dass der Dichtegradient-Lösung bei RT für die Zubereitung ist. </li><li> Zentrifuge bei 300 × g für 35 min bei RT ohne Bremse. Das Blut sollte in vier verschiedene Phasen trennen , wie in Figur 1A von oben nach unten gezeigt: Thrombozyten und Plasma, mononukleäre Zellen (white Ring), Dichtegradientenlösung, Granulozyten und Erythrozyten. </li><li> Sammeln Sie den weißen Ring, der die primären leukämischen Zellen mit einer Kunststoff-Pasteur-Pipette und an einem neuen 50-ml-Tube darstellt. <ol><li> Das Rohr wird mit PBS (enthält Calcium und Magnesium) bis zu 50 ml in total und Zentrifuge bei 300 g für 10 min bei RT. </li><li> bis zu 50 ml in total und Zentrifuge bei 300 g für 10 min bei RT das Pellet mit 5 ml PBS dann PBS hinzu. </li><li> Das Pellet mit komplettem RPMI-Medium (RPMI 1640 mit 10% fötalem Rinderserum (FBS), 2 mM Glutamin, 100 U / ml Penicillin und 100 mg / ml Streptomycin) zum Zählen der Lebensfähigkeit der Zellen-Zähler verwendet wird. </li></ol></li><li> Aspirieren die oberen Phasen der Granulozyten und Erythrozyten-Phase verlassen und PBS summieren sich zu 25 ml dann 3% Dextran in 0,9% NaClup in insgesamt 50 ml hinzu. Mischen Sie die Röhrchen 10 Mal und halten sie für 30 min bei RT ohne sich zu vermischen. </li><li> Sammeln Sie die obere RBC-armen Neutrophilen Schicht eind sie in ein sauberes steriles 50 ml Röhrchen platzieren und die Röhrchen bei 300 g für 10 min bei RT zentrifugiert. bis zu 50 ml insgesamt resuspendieren jedes Pellet mit 5 ml PBS dann Puffer Lyse der Erythrozyten hinzufügen. </li><li> Halten der Rohre in Dunkelheit für 15 min bei RT, dann bei 500 g für 10 min bei RT zentrifugiert. Waschen Sie die Pellets mit PBS (add PBS bis zu 50 ml insgesamt) bei 500 g zentrifugiert werden für 10 min bei RT. </li><li> Resuspendieren der Pellets mit komplettem RPMI-Medium zum Zählen der Lebensfähigkeit der Zellen Zähler verwendet wird. Halten Sie 3 x 10 5 jeder Zelltyp in 50 ul PBS-FBS (4%) in 5 ml Kunststoffröhrchen , die Reinheit ihrer Isolation zu bestimmen , mittels Durchflusszytometrie. </li></ol></li><li> Analysieren Sie die Morphologische Einsätze der isolierten Zellpopulationen <ol><li> Dazu resuspendieren je 3 x 10 4 Neutrophilen und 3 x 10 4 mononukleären Zellen in 150 &amp; mgr; l komplettes RPMI Medium. Montieren Sie die Glasobjektträger, Filterkarten und Probenkammern in der Cytospin Zentrifuge, um sicherzustellen, dass ter Zentrifuge ist gut ausbalanciert. </li><li> Platzieren Sie jede Zelltyp in die Probenkammer und Zyto-Zentrifuge bei 750 × g für 10 min bei RT Entfernen Sie die Folien, Filterkarten und Probenkammern aus der Zentrifuge. Disassemblieren vorsichtig, um nicht die Zellen auf dem Objektträger zu beschädigen. Entsorgen Filterkarten und Probenkammern. </li><li> Stain die Zellen mit Giemsa-Kit und halten Sie die Folien für 1 Stunde trocknen lassen. Untersuchen Sie die Zellen unter dem Mikroskop mit 100-facher Vergrößerung. </li></ol></li><li> Testen Sie die Reinheit der isolierten Zellen durch FACS <ol><li> Beschriften der isolierten primären leukämischen Zellen , die mit anti-human CD19 - Antikörper , konjugiert an APC (5 &amp; mgr; l / 10 6 Zellen). Beschriften der gereinigten Neutrophilen mit einer Mischung aus anti-human - Antikörper aufgelistet in Tabelle 1 (5 &amp; mgr; l / 10 6 Zellen). Inkubieren der Zellen in Dunkelheit für 30 min bei 4 o C. </li><li> Waschen Sie die Zellen mit 500 ul PBS-FBS (4%) der Zentrifuge die Röhrchen bei 300 g für 5 min bei RT. </li><li>Resuspendieren jedes Pellet in 200 &amp; mgr; l PBS-FBS (4%). </li><li> Analysieren Sie mit einem Durchflusszytometer, mit dem folgenden optischen Konfiguration: 488-nm-Laser: FSC-A (488 nm), SSC-A (488 / 10BP), FITC (530/30 BP), PerCP-Cy5.5 (695/40 BP ), PE (575/26 BP); 633-nm-Laser: APC (660/20 BP), APC-Cy7 (780/60 BP). Sicherstellen, dass die Farbkompensation. </li></ol></li><li> Cokultur von primären leukämischen Zellen mit Autologe Neutrophile <ol><li> Seed 2 x 10 5 Zellen / ml der primären leukämischen Zellen allein oder mit autologem Neutrophilen im Verhältnis 1:10 in komplettem RPMI - Medium. Dazu Zellzahlen durch Verdünnen von Zellsuspensionen einzustellen und mit 2 x 10 5 Zellen / ml der primären leukämischen Zellen zu 2 x 10 6 Zellen / ml Neutrophilen. Vorsichtig mischen. </li><li> Zugeben von 25 &amp; mgr; M Bruton-Tyrosinkinase (Btk) -Inhibitor (Ibrutinib) zu den Zellen. Inkubieren für 24 Stunden bei 37 ° C mit 5% CO 2. </li></ol></li><li> Zellernte und FACS Analyse <ol><li> Collect die Zellen in 5 ml Kunststoffröhrchen für die FACS-Analyse und zentrifugiert bei 300 g für 5 min bei RT die Rohre. Waschen der Pellets mit 1 ml PBS-FBS (4%), dann bei 300 g für 5 min bei RT zentrifugiert. </li><li> Resuspendieren Pellets in Annexin V und PI kommerziellen Kits und die Zellen in Dunkelheit für 10 min bei RT inkubiert. Analysieren Sie mit einem Durchflusszytometer die Gating - Strategie von 2A mit und der folgenden optischen Konfiguration: 488 - nm - Laser: FSC-A (488 nm), SSC-A (488 / 10BP), FITC (530/30 BP), PI (610 / 20 BP). Sicherstellen, dass die Farbkompensation. </li></ol></li></ol> 2. Differenzierung von HL60-Zellen entlang der Granulozytäre Pathway und ihre Cokultur mit RL-Lymphom B-Zellen in 3D-Modell <ol><li> Differenzierung von humanen promyelocytic (HL60) Zellen in Neutrophilen-ähnliche Zellen <ol><li> Einstellen HL60 Zellzahl bis 3 x 10 5 Zellen / ml fügen Sie dann 1 uM Retinsäure (RA) und 1,25% DMSO deren Differenzierung zu induzieren. Samen je 3 x 10 5 </sup&gt; HL 60 Zellen pro Vertiefung in 48-Well - Platte dann mit 5% CO 2 bei 37 ° C für 48 Stunden inkubiert. </li><li> Später wurden die Zellen und Zentrifuge bei 300 g für 5 min bei RT sammeln. Resuspendieren der Zellen mit komplettem RPMI-Medium zum Zählen der Lebensfähigkeit der Zellen Zähler verwendet wird. </li><li> Dann Zellsuspension in komplettem RPMI - Medium verdünnt auf HL60 Zellzahl bis 3 x 10 5 Zellen / ml und induzieren wieder ihre Differenzierung durch Zugabe von 1 &amp; mgr; M Retinsäure (RA) und 1,25% DMSO einzustellen. Samen je 3 x 10 5 HL60 - Zellen pro Well in 48-Well - Platte und Inkubation für weitere 48 Stunden bei 37 ° C mit 5% CO 2. </li></ol></li><li> Analyse von HL60 Differenzierung (HL60 diff) <ol><li> Sammeln Sie die Zellen und Zentrifugieren der Röhrchen bei 300 g für 5 min bei RT. Das Pellet mit komplettem RPMI-Medium zum Zählen der Lebensfähigkeit der Zellen Zähler verwendet wird. </li><li> Um die Änderungen in der Zelloberflächenmarker-Expression mittels Durchflusszytometrie analysiert. Legen Sie jedes 3 x 10 5 </sup&gt; undifferenzierte HL60 - Zellen (aus der Zellkultur) und 3 x 10 5 Zellen HL60 diff in 5 ml Kunststoffröhrchen für die FACS - Analyse und zentrifugieren die Röhrchen bei 300 g für 5 min bei RT. </li><li> Resuspendieren der Pellets in 50 &amp; mgr; l PBS-FBS (4%). Beschriften der Zellen mit anti-Mensch - CD11b konjugiert AF700 oder anti-human CD38 , konjugiert an APC (5 &amp; mgr; l / 10 6 Zellen). Inkubieren der Zellen in Dunkelheit für 30 min bei 4 o C. </li><li> Waschen mit 500 ul PBS-FBS (4%), dann bei 300 g für 5 min bei RT zentrifugiert. Resuspendieren der Pellets in 200 ul PBS-FBS (4%). </li><li> Analysieren Sie auf Durchflusszytometer mit der Gating - Strategie von 3A und die folgende optische Konfiguration: 488 - nm - Laser: FSC-A (488 nm), SSC-A (488 / 10BP); 633-nm-Laser: APC (660/20 BP), Alexa Fluor 700 (730/45 BP). </li><li> Um die morphologischen Veränderungen in HL60 diff zu testen, zu immobilisieren , die Zellen auf Glasobjektträger für die mikroskopische Untersuchung. <ol><li> Um dies zu tun, resuspendieren jedes 3 x 10 <sup&gt; 4 undifferenzierte HL60 - Zellen (aus Zellkultur) und 3 x 10 4 HL60 diff in 150 ul komplettem RPMI Medium. Montieren Sie die Glasobjektträger, Filterkarten und Probenkammern in der Cytospin Zentrifuge, um sicherzustellen, dass die Zentrifuge ausgeglichen ist. </li><li> Platzieren Sie jede Zelltyp in die Probenkammer und Zyto-Zentrifuge bei 1500 Umdrehungen pro Minute für 10 Minuten bei RT. Entfernen Sie die Folien, Filterkarten und Probenkammern aus der Zentrifuge. Disassemblieren vorsichtig, um nicht die Zellen auf dem Objektträger zu beschädigen. Entsorgen Filterkarten und Probenkammern. </li><li> Stain die Zellen mit Giemsa-Kit und halten Sie die Folien für 1 Stunde trocknen lassen. Untersuchen Sie die Zellen unter dem Mikroskop mit 100-facher Vergrößerung. </li></ol></li></ol></li><li> 3-dimensionale (3D) Kultur HINWEIS: Stellen Sie sicher, dass die Materialien, die in diesem Experiment verwendet werden, sind kalt und das Experiment auf Eis stattfindet. <ol><li> Resuspendieren je 5 x 10 4 RL - Zellen, entweder allein oder mit HL60 diff gemischt </em&gt; Im Verhältnis RL: HL60 diff 1:10 mit 300 Matrix ul Basalmembran 1 ml Pipettenspitze mit einer sterilen Schere , um die Öffnung zu etwa 2 bis 3 mm zu erweitern geschnitten werden. Vermeiden Blasen während dieses Schritts. </li><li> Samen je 300 ul Zellsuspension / Well in 24-Well-Platte. Vermeiden Blasen während dieses Schritts. Die Platte für 30 Minuten bei 37 ° C mit 5% CO 2 dann 1 addieren ml RPMI - Medium für jede Vertiefung und Inkubation für 7 Tage bei 37 ° C mit 5% CO 2 abzuschließen. Ändern Sie das Medium alle zwei Tage. In 10 nM Vincristin am Tag 5. </li></ol></li><li> FACS - Analyse <ol><li> Nach 7 Tagen Kultur absaugen Medium und waschen Sie jede Vertiefung mit 1 ml eiskaltem PBS zweimal. 3 ml / Vertiefung von eiskaltem PBS-EDTA (5 mM). Nehmen Sie das Gel aus dem Boden des Bohrlochs durch Abkratzen der Unterseite von 200 ul Pipettenspitze. Schütteln Sie die Platte vorsichtig auf Eis für 30 min. </li><li> Übertragen Zellsuspension in sterile 15-ml-Röhrchen und schütteln die Röhrchen sanft auf ice für weitere 30 min. Überprüfen Sie, ob das Auftreten von homogenen Zellsuspension. (Wenn es nicht der Fall ist, dann schütteln die Zellen für längere Zeit oder mehr hinzufügen PBS-EDTA). </li><li> Zentrifugiere die Röhrchen bei 300 xg für 10 min bei RT. Waschen Sie die Pellets mit PBS dann bei 300 g für 10 min bei RT zentrifugiert werden. </li><li> Resuspendieren mit PBS-FBS (4%) und Etikett mit anti-human - CD19 - Antikörper an PE-Cy7 konjugiert und Anti-Human - CD38 - Antikörper konjugiert an APC (5 &amp; mgr; l / 10 6 Zellen). In dunklen Inkubieren für 30 Minuten bei 4 ° C. </li><li> Waschen mit 500 ul PBS-FBS (4%), dann Zentrifugenröhrchen bei 300 g für 5 min bei RT. Resuspendieren der Zellen mit Annexin V und PI kommerziellen Kits. </li><li> Analysieren Sie auf Durchflusszytometer mit der Gating - Strategie von 4A und die folgende optische Konfiguration: 488 - nm - Laser: FSC-A (488 nm), SSC-A (488 / 10BP), FITC (530/30 BP), PI (610 / 20 BP), PE-Cy7 (780/60 BP); 633-nm-Laser: APC (660/20 BP). Sicherstellen, dass die Farbkompensation. </li></ol></li&gt; </ol>

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The authors have nothing to disclose.

Hier beschrieben, eine wirksame, einfach, schnell und preiswert Protokoll zur Isolierung von Neutrophilen aus menschlichem Blut mit hoher Reinheit Dichtegradientenzentrifugation Ansatzes und innerhalb der gleichen Schritt mononukleären Zellen werden ebenfalls abgetrennt und gewonnen. Die isolierten Zellpopulationen sind ≥90% rein. Es sind mehrere Verfahren für neutrophile Isolierung aus menschlichem Blut zur Verfügung. Diese umfassen ähnliche Verfahren unter Verwendung von diskontinuie...

Angeborene Immunzellen bilden einen wesentlichen Teil der Zellen innerhalb der Tumor - Mikroumgebung und wurden mit Tumor Malignität in Patienten und Tiermodellen von Krebs 1 verbunden. Vor kurzem hat es sehr geschätzt werden , dass eine chronische Immunreaktionen eine entscheidende Rolle bei der Förderung der Tumorprogression, Metastasierung und Resistenz gegen Chemotherapien 2 spielen. Makrophagen sind wichtige angeborenen Immunzellen , die direkt gezeigt worden ist, um Tumorzellantwort auf Chemotherapie 3,4 regulieren. Allerdings ist die Rolle der Neutrophilen, wichtige Akteure in der angeborenen Immunsystems, bei der Regulierung der Tumor Reaktion auf Anti-Krebs-Behandlung nicht bekannt. Die Ziele dieser Protokolle sind eine schnelle und glaubwürdige Methode zu verwenden Neutrophilen von CLL Patienten Blutproben zu trennen und HL60-Zellen entlang der granulocytic Weg, um ihre Rolle zu unterscheiden bei der Regulierung der Empfindlichkeit von Lymphomzellen zu Anti-Lymphom-Agenten zu studieren. 5 und wirken als erste Verteidigungslinie gegen Mikroorganismen 6 eindringen. Neutrophile haben eine wesentliche Rolle 7 wirksam angeborenen Immunantwort zusätzlich zu den variablen Effektor - Funktionen in verschiedenen pathologischen Zuständen in steigenden. Daher ist eine schnelle und glaubwürdige Methode Neutrophile von anderen Blutzellen, wie Dichtegradient - Trennverfahren zu isolieren, ist für in vitro - Studien erforderlich. Unter Verwendung dieses Verfahrens zur Isolierung von Neutrophilen erleichtert die weitere Forschung auf Neutrophilen-vermittelten immunologischen Funktionen in vivo und ex vivo. Die Fähigkeit , reine Populationen von Neutrophilen zu erhalten , ist ein wichtiger erster Schritt für die Untersuchung von Patienten mit immunologischen Erkrankungen 8. Dichtegradienten-Trennungsverfahren ist ein ideales Verfahren, bei dem eine hohe Ausbeute an Zellen erhalten wird. Die method beinhaltet die Zugabe von Dichtegradient-Lösung in den Boden eines Röhrchens verdünnten Humanblut durch Zentrifugation für 35 min bei 300 g ohne Bruch enthält, gefolgt. Der Ring der einkernigen Zellen erscheint an der Schnittstelle und die Neutrophilen liegen unterhalb des ersteren. Diese Verfahren haben erhebliche Vorteile gegenüber anderen verfügbaren Verfahren, wie Neutrophilen - Isolierungs - Kits , die 9 viel teurer sind. Zusätzlich Neutrophilen aus menschlichem Blut durch kommerzielle Kits unter Verwendung von Antikörpern, die an einen Oberflächenmarker spezifisch für humane Neutrophile, erhöhen das Risiko von Zell-Aktivierung oder Differenzierung zu isolieren. Dichtegradienten Trennungsverfahren erlaubt die Isolierung von neutrophilen Granulozyten innerhalb eines kurzen Zeitraums. Innerhalb der gleichen Schritt werden mononukleäre Zellen ebenfalls abgetrennt und gewonnen. Es ist eine grundlegende Technik, bei dem eine hohe Ausbeute an reinen Zellen, um erhaltene funktionelle Integrität zu erzielen. Um den Tumor microenv zu imitierenironment wurden 3D-Experimente durchgeführt. In Anbetracht der kurzen Halbwertszeit von Neutrophilen in vitro, sind die 3D - Experimente mit frischen menschlichen Neutrophilen nicht schlüssig. Dimethylsulfoxid (DMSO) und Retinsäure (RA) mit Hilfe der Differenzierungsinduktoren Aus diesem Grund promyelocytic (HL60-Zellen) induziert in Neutrophilen-ähnliche Zellen zu differenzieren. Mit differenzierten HL60 - Zellen (HL60 diff) wird verhindert , dass unterschiedliche Reaktionen von Neutrophilen, die aufgrund Isolierung von verschiedenen Spendern. In - vitro - 3D - Kulturmodelle stellen eine Zwischenstufe zwischen In - vitro - 2D - Modelle und in vivo Modellen. Im 2D-Kultur, breiteten sich die Zellen auf Kunststoff-Oberfläche unnatürliche Zelle Anhänge abgeschiedenen Proteine ​​bilden, die auf dieser synthetischen Oberfläche denaturiert werden. Umgekehrt können die Zellen in 3D-Kulturform natürliche Zell-Zell-Anlagen, da die Zellen und die extrazelluläre Matrix synthetisieren sie das natürliche Material, an dem sie angebracht sind.Aus diesem Grund 3D Kokultur Modellen, insbesondere zwischen Krebszellen und anderen Zelltypen, ist der Beitrag zu Tumorwachstum, Angiogenese und Metastasierung sehr nützlich für die Anzeige. Als Ergebnis machen 3D - Kulturen die Zellkultur , die physiologischen Bedingungen nachahmen , die mit 10 in vivo existieren.

<table border="0" cellpadding="0" cellspacing="0" width="1161">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">RPMI 1640&#160;</td>
			<td style="width:157px;">Gibco Invitrogen</td>
			<td style="width:156px;">21875-034</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fetal bovine serum (FBS)</td>
			<td>Gibco Invitrogen</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Phosphate-buffered saline (PBS contains calcium and magnesium)</td>
			<td>Gibco Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td>
			<td>Life technologies</td>
			<td style="width:156px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin (Pen Strep)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bruton&#39;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&#160;</td>
			<td style="width:157px;">CliniSciences</td>
			<td style="width:156px;">A3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vincristine&#160;</td>
			<td style="width:157px;">EG labo</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">BD Matrigel basement membrane matrix&#160;</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Put at 4 oC overnight before the day of the experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Red cell lysis buffer&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:156px;">555899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll (Pancoll)</td>
			<td>PAN Biotech</td>
			<td style="width:156px;">P04-60500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Retinoic acid</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">R2625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine serum albumin (BSA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>A7906</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">E5134</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sodium chloride (NaCl)</td>
			<td>Euromedex</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Annexing V-FLOUS staining kit&#160;</td>
			<td style="width:157px;">Roche</td>
			<td>11&#160;988&#160;549&#160;001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Kit RAL 555 Modified Giemsa staining kit</td>
			<td style="width:157px;">Cosmos Biomedical</td>
			<td style="width:156px;">CB361550-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRII flow cytometry</td>
			<td>BD Biosciences</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cytocentrifuge</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMR-XA microscope</td>
			<td style="width:157px;">Leica Microsystems</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cellometer Auto T4 Cell Viability Counter</td>
			<td style="width:157px;">Nexcelom&#160; Bioscience</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Dichtegradiententrennung hier beschriebenen Verfahren liefert primären leukämischen Zellen und unstimulierte aus dem Blut von CLL - Patienten isoliert Neutrophilen 1A die verschiedenen Blutschichten nach Dichtegradientenzentrifugation (von oben nach unten darstellt , die erhalten. Blutplättchen und Plasma, weißen Ring der einkernigen Zellen darstellt, Dichtegradientenlösung, Granulozyten und Erythrozyten). 1B und 1C zeigen die Unterschiede in den mo...

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

neutrophil-isolation-analysis-to-determine-their-role-lymphoma-cell

Research

JoVE Journal

Immunology and Infection

15.2K Aufrufe.  Cancer Research Center in Lyon. Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents. 

Serie: Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Ableitung von Thymuslymphom T-Zell-Linien aus Atm - / -</sup Und P53 - / -</sup Mäuse

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

Forschung

Immunologie und Infektion

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Isolierung von humanen Nabelschnur-Endothelzellen und ihre Verwendung in der Studie von neutrophilen Transmigration unter Strömungsbedingungen

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In-vitro-</em&gt; Assay auf Neutrophile Haftung auf Aktiviert primären humanen mikrovaskulären Endothelzellen unter statischen Bedingungen messen

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolierung humaner Monozyten durch Doppel-Zentrifugation und ihre Differenzierung zu Makrophagen in Teflon-beschichteten Zellkultur-Taschen

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Verfahren zur Inhibierung Bakterielle Pyomelanin Produktion und Bestimmen der entsprechenden Erhöhung der Empfindlichkeit gegenüber oxidativem Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

Methoden, um die regulatorische Rolle kleiner RNAs und Ribosomal Belegung von Untersuchen<em&gt; Plasmodium falciparum</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila der äußeren Membran - Vesikeln: Isolierung und Analyse ihrer Pro-inflammatorische Potential auf Makrophagen

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

In - vitro - Methoden zum Vergleich der Zielbindungs und CDC Induktion zwischen Therapeutische Antikörper: Anwendungen in Biosimilarity Analyse

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Überexpression und Reinigung des Menschen<em&gt; Cis</em&gt; -prenyltransferase in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Ein neuartiges Feeder-freie System für die Massenproduktion von murinen Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...