Wir danken Steve McBryant und Jeff Hansen für die Hilfe bei Ultrazentrifugation und Patti Kiser für die Hilfe bei der Maus Handhabung. Das Nationale Institut für neurologische Erkrankungen und Schlaganfall an den National Institutes of Health, gewähren 5R01NS056379-02 finanziert diese Arbeit.

<html> 1. Reinigung und Labeling Prion Rods Dieses Protokoll wird von einem zuvor veröffentlichten 18 angepasst <ol><li> Homogenisieren 100 Gramm Prion-infizierten Hirngewebe in 900 ml eiskaltem Homogenisierungspuffer (HB, 1X PBS mit 320 mM Saccharose, 150 mM NaCl und 4mm EDTA) 1 min bei maximaler Geschwindigkeit in einer kommerziellen Mixer, dann 2 min auf Eis. Dreimal wiederholen. </li><li> Centrifuge Homogenat 10 min bei 3000 xg und 4 ° C. Entfernen Sie und speichern Sie auf dem Eis Überstand. Re-suspend Pellet in 1L HB und wiederholen Sie die Schritte 1,1 und 1,2. </li><li> Pool Überstände und Zentrifuge für 60 min bei 100.000 xg, 0 ° C. Überstand. </li><li> Re-suspend Pellet in 100 ml 1X Tris-gepufferter Kochsalzlösung (10 mM Tris-HCl, 0,1 mM NaCl und 1 mM EDTA) mit 2% Triton X-100 (TBST). Estimate Proteinkonzentration nach der Bradford-oder ähnliche Tests und stellen bis 5 mg / ml in TBST. Gefühlt auf Eis für 30 min. </li><li> Zentrifuge für 30 min bei 100.000 xg, 0 ° C. Überstand verwerfen und Pellet waschen mit 50 ml TBST dann 100 mL TBS. Re-suspend Pellet in 100 ml 1X PBS mit 1% Sarcosyl und Protease-Inhibitor-Cocktail und rühren für 120 min bei 37 ° C. </li><li> Layer-Probe auf 900 ml 320 mM Saccharose und Zentrifuge für 60 min bei 100.000 xg, 10 ° C. Überstand verwerfen und wieder auszusetzen Pellet in 10 mL 2,3 M NaCl, 5% Sarcosyl. </li><li> Ultraschallbad Probe 5 x 40 Sekunden bei 70% eine Leistung von maximal 1 min Intervallen auf Eis. </li><li> Aliquot 1 mL Probe in Eppendorf-Röhrchen und zentrifugieren Sie für 15 Minuten bei 13.000 xg, 4 ° C. Wash Pellets zweimal mit TBS und speichern @ -70 ° C oder Konjugat in Schritt 1.9 Fluorochrom. </li><li> Konjugat 1 Tube gereinigt Prion Stangen mit 1 Fläschchen DyLight 649 Fluorochrom nach dem Protokoll des Herstellers. </li><li> Börse DyLight Kennzeichnung Puffer für PBS durch Dialyse oder Zentrifugation durch Filter Spalten. Shop in 20 ul Aliquots bei -70 ° C lichtgeschützt bis zur Verwendung. </li></ol> 2. Prion Impfung und die Erholung der Zellen Dieser Abschnitt umfasst intraperitoneal Prion Impfung und Gewinnung von Zellen aus dem Bauchfell, mediastinalen und mesenterialen Lymphknoten und der Milz. <ol><li> Verdünnen fluoreszierenden Prion Stangen 1:50 in PBS unmittelbar vor dem Gebrauch. Scruff Maus durch die dorsale Nackenfell mit Daumen und Zeigefinger sanft drehen Maus, Sie Gesicht und zurückhalten Schwanz mit kleinen Finger. Supinieren Maus, erhebend Hinterbeine Oberkörper über dem Kopf und hielt ihn leicht auf den Kopf. </li><li> Mit einer 30G Insulin-Spritze 100 ul von fluoreszierenden Prion Stäbe 1 cm lateral der Mittellinie und 1-2 cm vor dem Iliosakralgelenk. Führen Sie die Nadel 1 cm über die Haut gerichtet 45 ° nach medial. Verwenden 1:50-Verdünnung von fluoreszierenden Kügelchen oder PBS allein als Kontrolle. Inkubieren Mäusen vorgegebenen Zeit vor der Analyse. </li><li> Euthanize Mäusen nach anerkannten Tier in Pflege und Gebrauch Ausschuss-Protokolle. Injektion von 10 ml PBS in die Bauchhöhle mit einem 12-ml-Spritze und 25G-Nadel. Schütteln Sie Karkasse längs einer Minute. Recover Zellsuspension aus Peritoneum mit der gleichen Spritze und eine 16G Nadel in einem 15 mL konischen Rohr und Zurückhaltung auf Eis, während Karkasse Dissektion weiter. </li><li> Wet der Bauchseite der Karkasse gründlich mit 70% Ethanol. Heben Sie die Haut an der Mittellinie unterhalb des Brustkorbs mit einer Pinzette und schneiden Sie einen kleinen Schnitt durch die Haut mit einer Schere. Ab dort einen 3-5 cm langen Schnitt durch die Haut aus in beide Richtungen, um Kopf und Schwanz, dann zwei 2-cm Einschnitte von der Mittellinie seitlich von jedem Ende. Peel zurück und Pin die Haut um die Peritonealdialyse und Brusthöhle zu offenbaren. Vorsichtig durch die dünne transluzente Membranen umgeben das Bauchfell geschnitten und spiegeln den Brustkorb auf das Mediastinum zu offenbaren. </li><li> Suchen und entfernen Sie 2-4 Lymphknoten, leicht dorsal und seitlich neben dem Thymus, medial neben dem Truncus brachiocephalicus, in der Nähe der Bauchseite der Luftröhre. Suchen und entfernen Sie bis zu sechs mesenterialen Lymphknoten, in den weichen, weißen mesenterialen Gewebe eingebettet, die Anker der Darm mit der hinteren Bauchwand. Suchen und entfernen Sie die Milz, eine lange, dunkelrote Orgel befindet sich nur seitlich von der Mittellinie und dorsal in den Darm auf der linken Seite des Bauches. Reserve Lymphknoten in 1 ml RPMI 1640-Medium auf Eis, bis Dissektion ist abgeschlossen. </li><li> Mazerat und drücken Lymphknoten durch ein 40 um Sieb Sieb in 3 ml RPMI in einer Kunststoff-Petrischale mit dem Stempel einer 1 ml-Spritze. Spülen Sieb mit einer zusätzlichen 2 ml RPMI und übertragen Sie die 5 mL Zellsuspension in ein 15 ml konischen Rohr. Spülen Sie die Petrischale mit einer zusätzlichen 5 ml RPMI und Transfer zum gleichen Rohr. Zentrifuge 5 min bei 250 xg, Überstand verwerfen, resuspendieren Sie das Zellpellet in 1 ml FACS-Puffer (1X PBS, 1% Rinderserumalbumin und 10 mM EDTA) und in ein 1,5 ml Eppendorf-Röhrchen. </li></ol><html> ove_title &quot;&gt; 3. Zellfärbung und Durchflusszytometrie Das Folgende ist eine typische Färbung Protokoll zur Identifizierung von Immunzellen Teilmengen unter Verwendung von fluoreszierenden Antikörpern gegen gut charakterisierte Immunsystem Zelloberflächenmarker. Alle Antikörper wurden in FACS-Puffer unmittelbar vor der Verwendung verdünnt. <ol><li> Zählen von Zellen und aliquote 10 6 Zellen in Eppendorf-Röhrchen. Centrifuge Zellen (siehe 2.7) und waschen Pellet 2X mit 1 mL FACS-Puffer. </li><li> Inkubieren Zellen 20 min auf Eis in 100 ul von 2 ng / mL Ratte anti-Maus-CD16/32 endogene Fc-Rezeptoren zu blockieren. Pellet und waschen Sie die Zellen mit FACS-Puffer. </li><li> Add 100 L 1NG / mL geeigneten fluoreszierenden Antikörpern (y) n zu jeder Probe und Inkubation auf Eis für 1 Stunde. Geben Sie 100 ul FACS Puffer allein zu kontrollieren Zellen. Pellet und waschen Sie die Zellen zweimal mit FACS-Puffer. </li><li> Re-suspend-Zellen in 1 ml ACK-Puffer (150 mM NH 4 Cl, 1 mM KHCO 3 und 0,1 mM EDTA) auf Eis inkubieren für 1 min, um rote Blutkörperchen zu lysieren. Pellet und waschen Sie die Zellen mit FACS-Puffer. Re-suspend-Zellen in 1 ml FACS-Puffer. </li><li> Sammeln von Daten aus Zellen mit einem Durchflusszytometer der Lage, mehrfarbige Erkennung und Analyse, in der Regel mit 405, 488 und 633 nm Anregung Laser und fünf Minuten vor zehn Fluoreszenzemission Detektoren. </li></ol> 4. Repräsentative Ergebnisse: Wir reinigten Prion Stangen aus rohen, Prion-infizierten Hirnhomogenat (Abbildung 1) Waschmittel Solubilisierung und Ultrazentrifugation der E2 Elch Hirnhomogenat (Spuren 1 und 2) stark für Prion-Stäbchen (vergleiche Spuren 1 und 3), die auch Protease K angereichert resistent (Bahn 4). Interessanterweise angezeigt unverdaute gereinigt Prion Stäbe eine Glykoform Profil auffallend ähnlich verdaut E2 Hirnhomogenat, was darauf hinweist dramatischen Bereicherung für Prionen. Identisch behandelten normalen Hirnhomogenat produziert keine PK-resistente PrP C (Bahnen 5 und 6). Durchflusszytometrische Analyse von spezifischen Zellpopulationen zeigen, dass Antigen-präsentierende Zellen die Heimat des Bauchfells durch zwei Stunden und behalten fluoreszierende Kügelchen und Prion-Stäbchen, wie dramatisch erhöhte Fluoreszenz über PBS injiziert Kontrollen (Abbildung 2) belegt. Keine Zellen aus dem Peritoneum der PBS-behandelten Kontrollen angezeigt Fluoreszenz über dem Hintergrund (Abb. 2a-g) geerntet, während Zellen von Wulst (Abb. 2H-N) und Prionen (Abb. 2O-U)-inokulierten Mäuse zeigten signifikante Fluoreszenz, vor allem auf Monozyten (Abb. 2I und P), dendritische Zellen (Abb. 2 K und R) und Makrophagen (Abb. 2L und S), und einige Neutrophile (Abb. 2J und Q) und weniger B-Zellen (Abbildung 2T). Bead und Prion-tragenden Monozyten, dendritische Zellen und Makrophagen wurden auch in den Lymphknoten 2 Stunden nach der Impfung (Abb. 2AD und AK, AF und AM und AG und AN,), während nur wenige oder keine PrP CWD-tragenden Neutrophilen gefunden wurden B-oder T-Zellen gibt (Abb. 2AE und AL, AH und AG und AI und AP bezeichnet) gefunden. Wir erkannten keine Perlen oder Prionen-Stangen in der Milz oder mesenterialen Lymphknoten (Daten nicht gezeigt). In toto zeigen diese Daten, dass Immunzellen Verkehr sehr ähnlich Prionen auf andere partikuläre Antigene. <table border="1"><tbody><tr><td> REAGENT </td><td> Fluorochrom </td><td> Anregungslasers (nm) </td><td> Peakemission (nm) </td></tr><tr><td> Prion Stangen </td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1 um Perlen </td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> Antibodies </td><td></td><td></td><td></td></tr><tr><td> CD11b </td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td> Phycoerythrin-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11c </td><td> R-Phycoerythrin </td><td> 488 </td><td> 575 </td></tr><tr><td></td><td> Phycoerythrin-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R-Phycoerythrin </td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td> Allophycocyanin-Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R-Phycoerythrin </td><td> 488 </td><td> 575 </td></tr></tbody></table>&quot;&gt; Tabelle 1. Spektrale Eigenschaften von fluoreszierenden Reagenzien <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die Reinigung des Prion-Stäbchen aus infizierten Hirnhomogenat. Wir verwendeten eine 10% Rohprotein Hirnhomogenat von einem CWD-infizierter Rehe (E2, Bahnen 1 und 2) als Ausgangsmaterial, aus dem wir aggregierte Prion Stangen (Bahnen 3 und 4) gereinigt. Sowohl roh und gereinigt Materialien zeigte charakteristische Widerstand gegen Protease K-Behandlung (Spuren 1 bis 4), während die normalen Hirnhomogenat nicht (Bahnen 5 und 6). Molekulargewichtsmarker sind in Kd auf der linken Seite des Blots gezeigt. BH, Hirnhomogenat. <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Durchflusszytometrische Analyse von Immunzellen Menschenhandel Prionen aus dem Bauchfell an Lymphknoten 2 Stunden nach der Impfung. PBS (Platten AG und V-AB), wurden fluoreszierende Kügelchen (HN und AC-AI) oder Prion Stangen (OU und AJ-AP) injiziert in die Bauchhöhle von Mäusen und Zellen aus Peritoneallavage Flüssigkeit (AU) oder mediastinalen Lymphknoten (V-AP) Zwei Stunden später geerntet. Graphen in der ersten Spalte zeigen Zellen von Mäusen mit PBS (Abb. A und V), fluoreszierende Kügelchen (Panel H und AC) und Prion Stäbe (Tafeln A und AJ) behandelt. Fluoreszierende Zellen (rot oder grüne Punkte) und insgesamt Zellen (graue Punkte) aufgetragen, um die relative Größe (forward scatter), Granularität (side scatter) und Anteil der lebenden Zellen, die fluoreszieren zeigen. Eine erhebliche Anzahl von Zellen charakteristisch für Granulozyten erhalten fluoreszierende Kügelchen und Stäbchen. Die Zellen wurden auch mit Antikörpern gegen immune Zelloberflächenmarker und geschlossen für die LYG-Ly6C + Monozyten (Platten B, I, P, W, AD und AK), Ly6c-CD11c-CD11b + Ly6G + Neutrophile (C, J, Q, X, AE gebeizt und AL), Ly6G-Ly6C-CD11b + CD11c + dendritischen Zellen (D, K, R, Y, AF und AM), Ly6G-Ly6C-CD11c-CD11b + Makrophagen (E, L, S, Z, AG und AN), CD3 -B220 + CD21 + B-Zellen (F, M, T, AA, AH und AO) und CD21-B220-CD3 + T-Zellen (G, N, U, AB, AI und AP). Monozyten, dendritische Zellen und Makrophagen erhalten fluoreszierende Kügelchen und Prion-Stäbchen in die Bauchhöhle (Tafeln I und P, K und R und L bzw. S) und transportiert sie zu mediastinalen Lymphknoten (Platten AD und AK, AF und AM und AG und AN bezeichnet), zeigen ähnliche Aufnahme und den Handel mit diesen beiden Teilchen. Neutrophile erhalten eine erhebliche Menge von Kügelchen (J) und weniger Stangen (Q), scheiterte aber an ihnen zu den Lymphknoten zu liefern (AE und AL). B-Zellen erhalten und transportiert einige Stangen (T-und AO), aber praktisch keine Perlen (M und AH). T-Zellen Opfer weder Perlen noch Stangen (N, U, AI und AP).

<ol>
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Hier zeigen wir ein Protokoll für die Kennzeichnung und Nachverfolgung Prionen in vivo, dass erleichtert die Überwachung frühen Ereignisse in peripheren Prion-Infektion. Dieses Protokoll verbessert auf die bisherigen Versuche mit Überwachung Prion-Aufnahme in vitro 9 und in vivo 3,12 von pre-Kennzeichnung hochangereichertes Prion Inokulum eindeutig unterscheiden sie von endogenen PrP C. Da infektiöse Prionen und PrP C die gleiche primäre Aminos...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
</tr>
<tr>
<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

Vorhandensein einer anomalen Form eines Host-kodierten Prion-Protein (PrP), die Protease resistent, pathologische und ansteckend ist charakterisiert Prion-Erkrankungen wie das Chronic Wasting Disease (CWD) der Cerviden und Scrapie bei Schafen. Die Prion-Hypothese behauptet, dass dieses abnorme Konformer die meisten oder alle der infektiösen Prionen darstellt. Die Rolle des Immunsystems in der frühen Ereignisse in peripheren Prion Pathogenese konnte überzeugend für CWD und Scrapie 1-3 gezeigt. Transgene und pharmakologische Studien an Mäusen zeigten eine wichtige Rolle des Komplementsystems bei der Bindung und Replikation von Prionen früh nach der Infektion 4-6. In-vitro-und In-vivo-Studien haben auch Prionen Retention durch dendritische Zellen 7-10 beobachtet, obwohl ihre Rolle in Handel bleibt Unklar 11-16. Makrophagen sind ebenfalls in der frühen Prion Pathogenese in Verbindung gebracht, aber diese Studien haben sich auf Ereignisse, die Wochen nach der Infektion 3,11,17 konzentriert. Diese früheren Studien leiden auch unter dem Problem der Unterscheidung zwischen endogenen PrP C und infektiöse Prionen. Hier beschreiben wir eine semiquantitative, unvoreingenommene Herangehensweise für die Beurteilung der Prion-Aufnahme und-handel von der Impfstelle von Immunzellen rekrutiert dort. Aggregierte Prion Stäbe wurden aus infizierten Hirnhomogenat durch Waschmittel Solubilisierung von nicht aggregierten Proteinen und Ultrazentrifugation durch ein Saccharose-Kissen gereinigt. Polyacrylamid-Gelelektrophorese, Coomassie Blau-Färbung und Western Blot bestätigt Erholung von hoch angereichertem Prion Stangen in die pelletierten Fraktion. Prion Stangen wurden Fluorochrom-markierte dann intraperitoneal in Mäuse. Zwei Stunden später Immunzellen aus Peritoneallavage Flüssigkeit, Milz und mediastinalen und mesenterialen Lymphknoten wurden für Prion Stabrückhaltesystem und Zell-Untergruppen von multicolor Durchflusszytometrie unter Verwendung von Markern für Monozyten, Neutrophile, dendritische Zellen, Makrophagen und B-und T-Zellen identifiziert untersucht. Dieser Test ermöglicht es zum ersten Mal die direkte Überwachung der Immunzellen Erwerb und Handel mit Prionen in vivo innerhalb von Stunden nach der Infektion. Dieser Assay auch deutlich unterscheidet infektiöse, aggregiert Prionen aus PrPC in der Regel auf dem Host-Zellen exprimiert, die als schwierig und führt zur Interpretation der Daten Probleme in anderen Testsystemen können. Dieses Protokoll kann auch auf andere Impfung Routen (orale, intravenöse, intranervous und subkutanen, zB) und Antigene (konjugierte Beads, bakteriellen, viralen und parasitären Erregern und Proteine, Ei) als auch angepasst werden.

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Hier beschreiben wir eine neuartige Assays für die Überwachung der Prion-Aufnahme und-handel durch Immunzellen sofort nach intraperitonealer Inokulation durch Reinigung und Fluoreszenzmarkierung aggregierte Prion Stangen aus infizierten Gehirn Material dann die Überwachung ihrer Aufnahme und Bewegung an der Injektionsstelle und Charakterisierung der Zellen Vermittlung dieser Ereignisse.

Überwachung Immunzellen Trafficking Fluorescent Prion Rods Stunden nach intraperitonealer Infektion

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K Aufrufe.  Colorado State University. Hier beschreiben wir eine neuartige Assays für die Überwachung der Prion-Aufnahme und-handel durch Immunzellen sofort nach intraperitonealer Inokulation durch Reinigung und Fluoreszenzmarkierung aggregierte Prion Stangen aus infizierten Gehirn Material dann die Überwachung ihrer Aufnahme und Bewegung an der Injektionsstelle und Charakterisierung der Zellen Vermittlung dieser Ereignisse.

Serie: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Forschung

Immunologie und Infektion

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Seven Steps to Cells Stellate

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolierung von Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Durchflusszytometrische Isolation of Primary Murine Type II Alveolarepithelzellen für Funktionelle und Molekulare Studien

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolation von Fettgewebe Immune Cells

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Generierung von Lymphknoten-Fett-Pad Chimären für die Studie der Lymphknoten Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Detektion von fluoreszierenden Nanopartikel Wechselwirkungen mit primären Immun-Zell-Subpopulationen durch Durchflusszytometrie

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Ein Semi-automatisierten Ansatz zur Vorbereitung Antikörper-Cocktails für immunphänotypische Analyse von menschlichem peripherem Blut

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Zuverlässig und High Efficiency Extraktion von Nieren-Immunzellen

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...