Nous remercions Steve et Jeff Hansen McBryant de l&#39;aide pour l&#39;ultracentrifugation et Patti Kiser de l&#39;aide pour la manipulation de la souris. L&#39;Institut national des maladies neurologiques et des maladies au National Institutes of Health, accorder 5R01NS056379-02 financé ce travail.

<html> 1. Purifiant et l&#39;étiquetage des tiges à prions Ce protocole est adapté d&#39;un 18 précédemment publiés <ol><li> Homogénéiser 100 grammes de prion infectées par le tissu cérébral dans 900 ml de glace de tampon d&#39;homogénéisation froid (HB, 1X PBS contenant 320 mM de saccharose, NaCl 150 mM EDTA et 4mm) 1 min à vitesse maximale dans une publicité mixeur, puis 2 min sur la glace. Répétez trois fois. </li><li> Centrifuger l&#39;homogénat pendant 10 min à 3000 xg et 4 ° C. Retirer et conserver le surnageant sur la glace. Re-suspendre le culot dans 1L HB et répétez les étapes 1.1 et 1.2. </li><li> Surnageants Piscine et centrifuger pendant 60 min à 100 000 xg, à 0 ° C. Rejeter le surnageant. </li><li> Re-suspendre le culot dans 100 ml d&#39;une solution saline tamponnée Tris 1X (10 mM Tris-HCl, 0,1 mM de NaCl et 1 mM EDTA) contenant 2% de Triton X-100 (TBST). Estimer la concentration de protéines par les Bradford ou dosage similaire et ajuster à 5 mg / mL dans TBST. Réfrigérer la glace pendant 30 min. </li><li> Centrifuger pendant 30 min à 100 000 xg, à 0 ° C. Rejeter le surnageant et laver culot avec 50 ml TBST puis 100 ml SCT. Re-suspendre le culot dans 100 ml PBS 1X contenant sarcosyl 1% et le cocktail inhibiteur de la protéase et remuer pendant 120 min à 37 ° C. </li><li> Couche échantillon sur 900 ml de 320 mM de saccharose et centrifuger pendant 60 min à 100 000 xg, 10 ° C. Rejeter le surnageant et remettre en suspension le culot dans 10 ml de NaCl 2,3 M, sarcosyl 5%. </li><li> Soniquer échantillon de 5 x 40 secondes à puissance maximale de 70% à intervalles de 1 min sur la glace. </li><li> Aliquote de 1 ml d&#39;échantillon dans des tubes Eppendorf et centrifuger pendant quinze minutes à 13000 xg, 4 ° C. Lavez granules deux fois avec le SCT et stocker @ -70 ° C ou conjugué au fluorochrome à l&#39;étape 1.9. </li><li> Conjugué 1 tube de cannes à prions purifiés en utilisant une fiole de 649 DyLight fluorochrome selon le protocole du fabricant. </li><li> Échange de tampon étiquetage DyLight pour PBS par dialyse ou par centrifugation à travers des colonnes de filtration. Boutique en 20 aliquotes à -70 ° C loin de la lumière jusqu&#39;au moment de servir. </li></ol> 2. L&#39;inoculation de prions et la récupération des cellules Cette section englobe l&#39;inoculation du prion par voie intrapéritonéale et la récupération des cellules du péritoine, les ganglions lymphatiques médiastinaux et mésentériques et la rate. <ol><li> Diluer tiges prion fluorescentes 1:50 dans du PBS immédiatement avant l&#39;utilisation. La souris Scruff par la fourrure dorsale du cou avec le pouce et l&#39;index, tourner doucement la souris face à vous et à freiner la queue avec petit doigt. Supination souris, élevant le torse arrière dessus de la tête et lui tenant légèrement à l&#39;envers. </li><li> En utilisant une seringue d&#39;insuline 30G, injecter 100 ml de tiges prion fluorescent 1 cm de la ligne médiane et 1-2 cm en avant de l&#39;articulation sacro-iliaque. Insérez l&#39;aiguille à 1 cm par la peau réalisé à 45 ° en dedans. Utilisez dilution 1:50 de perles fluorescentes ou PBS seul comme témoins. Incuber souris pour le temps imparti avant l&#39;analyse. </li><li> Euthanasier les souris en fonction de soins des animaux et l&#39;utilisation de protocoles approuvés comité. Injecter 10 ml de PBS dans la cavité péritonéale utilisant une seringue de 12 ml et une aiguille 25G. Secouez délicatement la carcasse longitudinalement pendant une minute. Récupérer suspension de cellules provenant du péritoine en utilisant la même seringue et une aiguille 16G dans un tube de 15 ml conique et réserve sur la glace tout en la dissection des carcasses se poursuit. </li><li> Mouiller la partie ventrale de la carcasse soigneusement avec de l&#39;éthanol à 70%. Soulever la peau au niveau de la ligne médiane, juste sous la cage thoracique avec une pince et couper une petite incision dans la peau avec des ciseaux. À partir de là, faire une incision 3-5 cm de long à travers la peau dans les deux directions à partir de la tête et la queue, puis deux incisions de 2 cm de la ligne médiane latéralement de chaque côté. Peler et la broche de la peau pour révéler les cavités péritonéale et thoracique. Découpez soigneusement à travers les membranes minces translucides entourant le péritoine et reflètent le thorax pour révéler le médiastin. </li><li> Trouver et supprimer 2-4 ganglions lymphatiques médiastinaux, légèrement dorsale et latérale adjacente au thymus, médiane adjacente à l&#39;artère brachiocéphalique, près de la face ventrale de la trachée. Trouver et supprimer jusqu&#39;à six ganglions mésentériques, intégrés dans le tissu mésentérique blanc doux qui ancre les intestins pour la paroi abdominale postérieure. Trouver et supprimer la rate, un long, orgue rouge foncé situé juste latéral de la ligne médiane et dorsale de l&#39;intestin sur le côté gauche de l&#39;abdomen. Ganglions lymphatiques réserve dans 1 ml de milieu RPMI 1640 sur la glace jusqu&#39;à ce que la dissection est terminée. </li><li> Laisser macérer et appuyez sur les ganglions lymphatiques à travers une passoire 40 um dans 3 mL de RPMI dans un plat en plastique de Petri en utilisant le piston d&#39;une seringue de 1 ml. Rincer la crépine avec un supplément de 2 ml de RPMI et le transfert de la suspension 5 ml de cellules dans un tube de 15 ml conique. Rincez les boîtes de Pétri avec un supplément de 5 ml de RPMI et le transfert dans le même tube. Centrifuger 5 min à 250 xg, éliminer le surnageant, remettre en suspension le culot cellulaire dans 1 ml de tampon FACS (PBS 1X, 1% d&#39;albumine sérique bovine et 10 mM d&#39;EDTA) et transférer dans un tube Eppendorf de 1,5 ml. </li></ol><html> ove_title &quot;&gt; 3. Cellule de coloration et de cytométrie en flux Ce qui suit est un protocole de coloration typique pour l&#39;identification des sous-ensembles de cellules immunitaires utilisant des anticorps fluorescents contre le bien-immunes caractérisées marqueurs de surface cellulaire. Tous les anticorps ont été dilués dans du tampon FACS immédiatement avant utilisation. <ol><li> Comptez cellules et 6 cellules aliquote de 10 dans des tubes Eppendorf. Centrifuger les cellules (voir 2.7) et laver granules 2X avec 1 ml de tampon FACS. </li><li> Incuber les cellules 20 min sur la glace dans 100 ul de 2 ng / mL chez le rat anti-souris CD16/32 pour bloquer les récepteurs Fc endogène. Pellet et les cellules de lavage avec du tampon FACS. </li><li> Ajouter 100 ul de 1 ng / mL appropriée fluorescentes antibod (nt) à chaque échantillon et incuber sur de la glace pendant une heure. Ajouter 100 ul de tampon FACS seul pour les cellules de contrôle. Pellet et cellules laver deux fois avec du tampon FACS. </li><li> Re-suspendre les cellules dans une mémoire tampon ACK ml (150 mM de NH 4 Cl, 1 mM de KHCO3 et 0,1 mM d&#39;EDTA) Incuber dans la glace pendant 1 min pour lyser les globules rouges. Pellet et les cellules de lavage avec du tampon FACS. Re-suspendre les cellules dans 1 ml de tampon FACS. </li><li> Recueillir des données à partir des cellules en utilisant un cytomètre en flux capable de détecter multicolore et d&#39;analyse, généralement avec des lasers 405, 488 et 633 nm d&#39;excitation et de cinq à dix détecteurs d&#39;émission de fluorescence. </li></ol> 4. Les résultats représentatifs: Nous avons purifié à partir des tiges prion brut, solubilisation Détergent prion homogénat de cerveau infecté (figure 1) et d&#39;ultracentrifugation de l&#39;homogénat E2 élans du cerveau (voies 1 et 2) considérablement enrichi pour les tiges de prion (comparer les pistes 1 et 3), qui était également la protéase K résistants (piste 4). Fait intéressant, non digérées tiges prions purifiés affiché un profil glycoforme étonnamment semblables à E2 homogénat cérébral digéré, indiquant un enrichissement spectaculaire des prions. Identiquement traités homogénat de cerveau normal produit aucun PK-résistante PrP C (pistes 5 et 6). L&#39;analyse par cytométrie en flux des populations de cellules spécifiques de démontrer que les cellules présentatrices d&#39;antigènes chez le péritoine de deux heures et de retenir des billes fluorescentes et les tiges à prion, comme en témoigne par fluorescence considérablement augmenté au cours des contrôles PBS injecté (figure 2). Pas de cellules récoltées dans le péritoine de PBS témoins traités affiché fluorescence au-dessus de fond (fig. 2A-G), tandis que les cellules du cordon (fig. 2H-N) et du prion (fig. 2O-U)-souris inoculées affiché par fluorescence significative, en particulier sur les monocytes (fig. 2I et P), les cellules dendritiques (fig. 2K et R) et les macrophages (fig. 2L et S), et quelques neutrophiles (figure 2J et Q) et les cellules B moins (2T figure). Perle et prions portant monocytes, cellules dendritiques et les macrophages ont également été trouvés dans les ganglions lymphatiques médiastinaux deux heures après l&#39;inoculation (fig. 2AD et AK, AF et AM et AG et AN, respectivement), tandis que peu ou pas de PrP CWD portant neutrophiles , les cellules B ou T ont été trouvés là (fig. 2AE et AL, AH et AG et AI et AP, respectivement). Nous n&#39;avons détecté aucune perles ou des baguettes de prion dans la rate ou des ganglions mésentériques (données non présentées). Dans sa totalité, ces données démontrent que la circulation des cellules immunitaires prions très similaire à des antigènes autres particules. <table border="1"><tbody><tr><td> REACTIFS </td><td> Fluorochrome </td><td> Excitation laser (nm) </td><td> Pic d&#39;émission (nm) </td></tr><tr><td> Tringles à prions </td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> Billes 1 um </td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> Anticorps </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> Phycoérythrine-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11c </td><td> R-phycoérythrine </td><td> 488 </td><td> 575 </td></tr><tr><td></td><td> Phycoérythrine-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-phycoérythrine </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> Allophycocyanine-Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R-phycoérythrine </td><td> 488 </td><td> 575 </td></tr></tbody></table>»&gt; Tableau 1. Propriétés spectrales des réactifs fluorescents <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="Figure 1" /> Figure 1. Purification des tiges prions d&#39;homogénat de cerveau infecté. Nous avons utilisé un homogénat brut de 10% du cerveau d&#39;un cerf MDC-infectés (E2, les voies 1 et 2) comme matériau de départ à partir de laquelle nous avons purifié tiges prion agrégées (voies 3 et 4). Les deux matériaux bruts et purifiés ont montré une résistance caractéristique à la protéase K traitement (voies 1 - 4), tandis que homogénat de cerveau normal ne (pistes 5 et 6). Des marqueurs de poids moléculaire sont présentés dans Kd à la gauche de la tache. BH, homogénat de cerveau. <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="Figure 2" /> Figure 2. L&#39;analyse par cytométrie en flux de cellules immunitaires prions le trafic du péritoine vers les ganglions lymphatiques médiastinaux deux heures après l&#39;inoculation. PBS (panneaux AG et V-AB), des perles fluorescentes (HN et AC-AI) ou de barres à prions (UO et AJ-AP) ont été injectée dans le péritoine des souris et des cellules récoltées à partir du liquide de lavage péritonéal (UA) ou les ganglions lymphatiques médiastinaux (V-AP), deux heures plus tard. Les graphiques dans les premières cellules montrent colonne à partir de souris traitées avec du PBS (panneaux A et V), des perles fluorescentes (panneau H et AC) et les tiges du prion (panneaux O et AJ). Cellules fluorescentes (points rouges ou vertes) et des cellules totales (points gris) sont tracées pour montrer la taille relative (diffusion vers l&#39;avant), la granularité (diffusion latérale) et la proportion du total des cellules vivantes qui sont fluorescents. Un nombre important de cellules caractéristiques des granulocytes conservé perles fluorescentes et les tiges. Les cellules ont également été colorées avec des anticorps contre le système immunitaire des marqueurs de surface cellulaire et fermé pour LYG-Ly6C + monocytes (panneaux B, I, P, W, AD et AK), Ly6c-CD11c-CD11b + Ly6G + neutrophiles (C, J, Q, X, AE et AL), Ly6G-Ly6C-CD11b + CD11c + cellules dendritiques (D, K, R, Y, AF et AM), Ly6G-Ly6C-CD11c-CD11b + macrophages, (E, L, S, Z, AG et AN), CD3 -B220 + CD21 + cellules B (F, M, T, AA, AH et AO) et CD21-B220-cellules T CD3 + (G, N, U, AB, AI et AP). Les monocytes, les cellules dendritiques et les macrophages conservé perles fluorescentes et les tiges du prion dans la cavité péritonéale (panneaux I et P, K et R et L et S, respectivement) et les a transportés vers les ganglions lymphatiques médiastinaux (panneaux AD et AK, AF et AM et AG et AN, respectivement), démontrant ainsi l&#39;absorption similaires et la traite de ces deux particules. Les neutrophiles conservé une importante quantité de perles (J) et moins de tiges (Q), mais a omis de les livrer aux ganglions lymphatiques (AE et AL). Les cellules B conservé et transporté quelque tiges (T et AO), mais pratiquement pas de perles (M et H). Les cellules T, ni victimes, ni perles baguettes (N, U, AI et AP).

<ol>
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Ici nous démontrons un protocole de marquage et de suivi des prions in vivo qui facilite grandement le suivi des événements précoces de l&#39;infection à prion périphériques. Ce protocole améliore grandement sur ​​les tentatives passées au absorption prion surveillance in vitro et in vivo 9 3,12 en pré-étiquetage inoculum prion hautement enrichi sans ambiguïté le différencier de la PrP C endogène. Parce que les prions infectieux et la PrP C p...

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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

Présence d&#39;une forme anormale d&#39;une protéine prion codée par l&#39;hôte (PrPc) qui est résistant à la protéase, pathologiques et infectieux caractérise les maladies à prions comme la maladie débilitante chronique (MDC) des cervidés et la tremblante du mouton. L&#39;hypothèse du prion, affirme que ce conformère anormal constitue plupart ou la totalité du prion infectieux. Le rôle du système immunitaire dans les événements précoces dans la pathogenèse du prion périphérique a été démontré de façon convaincante pour le MDC et la tremblante 1-3. Des études pharmacologiques transgéniques et des souris ont révélé un rôle important du système du complément à conserver et reproduire les prions tôt après l&#39;infection 4-6. In vitro et in vivo ont également observé la rétention des prions par les cellules dendritiques 7-10, bien que leur rôle dans le trafic reste clair 11-16. Les macrophages ont eux aussi été impliqués dans la pathogenèse du prion début, mais ces études ont porté sur les événements survenus semaines après l&#39;infection 3,11,17. Ces études antérieures souffrent également du problème de la différenciation entre endogènes PrP C et les prions infectieux. Nous décrivons ici un semi-quantitative, approche impartiale pour évaluer l&#39;absorption du prion et le trafic du site d&#39;inoculation par les cellules immunitaires recrutées là. Tringles à prions ont été agrégées purifiée à partir de l&#39;homogénat de cerveau infecté par solubilisation d&#39;un détergent non agrégées de protéines et d&#39;ultracentrifugation sur un coussin de saccharose. Électrophorèse sur gel polyacrylamide, le bleu de Coomassie coloration et l&#39;ouest de la récupération buvard confirmés de tiges prion hautement enrichi dans la fraction culot. Tringles à prions ont été marqués par voie intrapéritonéale fluorochrome, puis injectées dans des souris. Deux heures plus tard les cellules immunitaires du liquide de lavage péritonéal, la rate et les ganglions lymphatiques médiastinaux et mésentériques ont été analysés pour la rétention des prions tige et sous-ensembles de cellules identifiées par cytométrie en flux utilisant des marqueurs multicolores pour les monocytes, les neutrophiles, les cellules dendritiques, les macrophages et les cellules B et T. Ce test permet la surveillance directe de la première fois des cellules immunitaires acquisition et prions le trafic in vivo dans les heures suivant l&#39;infection. Ce test a également différencie clairement infectieuses, les prions agrégés à partir de la PrPC normalement exprimé sur les cellules hôtes, ce qui peut être difficile et conduire à des problèmes d&#39;interprétation des données dans les systèmes de dosage d&#39;autres. Ce protocole peut être adapté à d&#39;autres voies d&#39;inoculation (voie orale, intraveineuse, intranervous et sous-cutanée, par exemple) et les antigènes (perles conjugués, les agents pathogènes bactériens, viraux et parasitaires et les protéines, les œufs) ainsi.

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

Nous décrivons ici un nouveau test pour la surveillance et le trafic de l&#39;absorption du prion par les cellules immunitaires, immédiatement après l&#39;inoculation intrapéritonéale en purifiant et en fluorescence étiquetage tiges prion agrégés à partir de tissu cérébral infecté, puis le suivi de leur absorption et le mouvement à partir du site d&#39;injection et la caractérisation des cellules médiation de ces événements.

Surveillance des cellules immunitaires trafic fluorescent Rods Prion heures après l&#39;infection par voie intrapéritonéale

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 vues.  Colorado State University. Nous décrivons ici un nouveau test pour la surveillance et le trafic de l'absorption du prion par les cellules immunitaires, immédiatement après l'inoculation intrapéritonéale en purifiant et en fluorescence étiquetage tiges prion agrégés à partir de tissu cérébral infecté, puis le suivi de leur absorption et le mouvement à partir du site d'injection et la caractérisation des cellules médiation de ces événements.

Vidéo: 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.

Mesure de l&#39;activité calpaïne dans des cellules fixées et de la Vie par cytométrie en flux

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

Recherche

Immunologie et infection

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.

Sept étapes pour Stellate Cellules

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

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Débit analyse par cytométrie de cellules immunitaires dans aortes murin

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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.

Isolement des cellules immunitaires fonctionnelles cardiaques

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.

Après Cell-sort<em&gt; E. coli</em&gt; Après infection par le 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 ...

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.

Isolation par cytométrie en flux des cellules primaires murins épithéliales alvéolaires de type II pour les études fonctionnelles et moléculaires

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.

Isolement de tissu adipeux cellules immunitaires

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.

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.

Détection de nanoparticules fluorescentes Interactions avec immunitaire primaire sous-populations de cellules par cytométrie en flux

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.

Une approche semi-automatique pour la préparation des anticorps Cocktails pour immunophénotypique Analyse de sang périphérique humain

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