Name: harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles
Uploaded: 2012-06-08T12:00:00
Description: Watch this Scientific Journal Video about Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles at JoVE.com

Yazarlar için Iowa State University Akım Sitometri Tesisi mali destek ve Dr Shawn Rigby için ABD Ordusu Tıbbi Araştırma ve Malzeme Komutanlığı (Grant Numaraları W81XWH-09-1-0386 ve W81XWH-10-1-0806) teşekkür etmek istiyorum Onun uzman teknik yardım.

1. Akciğer Lavaj kullanarak Mouse Alveoler Makrofajlar (AMɸ) Hasat <ol><li> Böyle bir laboratuvar önlüğü, tek kullanımlık eldivenler, ve uygun göz koruması gibi uygun kişisel koruyucu ekipman (PPE) giyiniz. </li><li> Hasat başlamadan önce tam AMɸ (cAMɸ) orta hazırlayın. Penisilin / streptomisin Dulbelcco en Modified Eagle Medium (DMEM) ile 222,25 mL (kalem / Strep) çözeltisi, β-merkaptoetanol, 250 uL (2-merkaptoetanol), ve ısı inaktive edilmiş fetal bovin serumu (FBS) ve 25 ml 2.5 ml ilave edil...

<ol>
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Tek doz rejimleri 5 intranazal uygulandığında polianhidrid nanoparçacık aşı platformları etkinliğini göstermiştir. Bu aşı dağıtım platformu tarafından uyarılan akciğerlerde ikamet fagositik hücre popülasyonlarının aktivasyon Ölçme potansiyel yeteneğinin değerlendirilmesi sonuçta adaptif bağışıklık yanıtları teşvik etmek için izin verir. Özellikle akciğer lavaj sıvısında makrofaj hasat ve nanopartiküllerin farklı formülasyonları ile onl...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> CAM Medya </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> 50 mM 2-merkaptoetanol </td><td> Sigma </td><td> M3148-25ml </td><td></td></tr><tr><td> Penisilin / streptomisin 10,000 ug / ml solüsyon </td><td> Cellgro </td><td> 30-002-CI </td><td></td></tr><tr><td> Sığır fetüs serumu </td><td> Atlanta Biologicals </td><td> S11150 </td><td></td></tr><tr><td> FACS Tampon </td><td></td><td></td><td></td></tr><tr><td> Sodyum klorür </td><td> Fisher Scientific </td><td> S671-500 </td><td></td></tr><tr><td> Sodyum fosfat </td><td> Fisher Scientific </td><td> MK7868500 </td><td></td></tr><tr><td> Potasyum klorür </td><td> Fisher Scientific </td><td> P217500 </td><td></td></tr><tr><td> Potasyum fosfat </td><td> Fisher Scientific </td><td> P288-200 </td><td></td></tr><tr><td> BSA (Bovine Serum Albumin) </td><td> Sigma </td><td> A7888 </td><td></td></tr><tr><td> Sodyum Azid </td><td> Sigma </td><td> S2002 </td><td></td></tr><tr><td> Antikorlar </td><td></td><td></td><td></td></tr><tr><td> Rat IgG </td><td> Sigma </td><td> I4341 </td><td></td></tr><tr><td> Anti-Ms CD16/32 </td><td> eBioscience </td><td> 16-0161 </td><td></td></tr><tr><td> Anti-Ms MHC II haplotip floresein izotiyosiyanat (FITC) IA / IE, klon M5/114.15.2, konjuge </td><td> eBioscience </td><td> 11-5321 </td><td></td></tr><tr><td> Fare anti-CD86, allophycocyanin ile konjuge klonu GL-1, (APC)-Cy7 </td><td> Biolegend </td><td> 105030 </td><td></td></tr><tr><td> APC konjuge anti-fare CD40, klon 1C10, </td><td> eBioscience </td><td> 17-0401 </td><td></td></tr><tr><td> Biotin için konjuge anti-fare CD209, klon 5H10, </td><td> eBioscience </td><td> 13-2091 </td><td></td></tr><tr><td> Alexa Fluor 700 konjuge anti-fare CD11b, klon M1/70, </td><td> eBioscience </td><td> 56-0112 </td><td></td></tr><tr><td> Phycoerythrin ile konjuge edilmiş anti-fare F4/80, klon BM8, (PE)-Cy7 </td><td> eBioscience </td><td> 25-4801 </td><td></td></tr><tr><td> PE-Texas kırmızı konjuge Streptavidin </td><td> BD Biosciences </td><td> 551487 </td><td></td></tr><tr><td> Diğer Sarf Malzemeleri ve Reaktifler </td><td></td><td></td><td></td></tr><tr><td> Etanol </td><td> Fisher Scientific </td><td> A405-20 </td><td> 70% (v / v) olarak kullanıldı </td></tr><tr><td> Sıkıştırılmış CO 2 </td><td> Linweld </td><td> 16000060 </td><td></td></tr><tr><td> 1 ml Şırınga </td><td> BD Biosciences </td><td> 309659 </td><td></td></tr><tr><td> Egemen 3 ½ &quot;Fr Tom Catcatheter </td><td> Kendall </td><td> 703021 </td><td></td></tr><tr><td> Biyogüvenlik Kabini </td><td> NUAIRE </td><td> Series 22 </td><td></td></tr><tr><td> Diseksiyon Makas </td><td> Balıkçı </td><td> 138082 </td><td></td></tr><tr><td> Forseps </td><td> Roboz </td><td> RS-8254 </td><td></td></tr><tr><td> PBS, kalsiyum ve magnezyum içermeyen 1X </td><td> Cellgro </td><td> 21-040-CM </td><td></td></tr><tr><td> Vida Kapaklı 15 ml Santrifüj Tüpleri </td><td> VWR International </td><td> 21008-216 </td><td></td></tr><tr><td> Altı sıra Doku Kültürü Tabaklar Tedavi </td><td> Costar </td><td> 3516 </td><td></td></tr><tr><td> Plastik Boru Raflar </td><td> Nalgene </td><td> 5970 </td><td></td></tr><tr><td> Hücre Kazıyıcı 24 cm </td><td> DYP </td><td> 99002 </td><td></td></tr><tr><td> 5 ml Polistiren Yuvarlak Alt Tüp </td><td> Şahin </td><td> 352008 </td><td></td></tr><tr><td> Pipetleyin yardım XL </td><td> Drummond </td><td> 4-000-105 </td><td></td></tr><tr><td> 10, 5, ve 2 mL Pipetler </td><td> Balıkçı </td><td> 13-675 </td><td></td></tr><tr><td> 200 ve 10 uL micropipettors </td><td> Gilson Pipetman </td><td> F123601 </td><td></td></tr><tr><td> 200 ve 10 uL pipet uçları </td><td> Balıkçı </td><td> 02-707 </td><td></td></tr><tr><td> BD DengeleyiciSabitleştirici </td><td> BD Biosciences </td><td> 338036 </td><td></td></tr><tr><td> ISOTON II Dilüent </td><td> Beckman-Coulter </td><td> 8546719 </td><td></td></tr><tr><td> Zap-oglobin II Parçalayıcı Reaktif </td><td> Beckman-Coulter </td><td> 7546138 </td><td></td></tr><tr><td> Coulter Sayaç Polistiren Flakon </td><td> Beckman-Coulter </td><td> 14310-684 </td><td></td></tr><tr><td> Test Tüpleri </td><td> BD Biosciences </td><td> 352008 </td><td></td></tr><tr><td> Ekipman </td><td></td><td></td><td></td></tr><tr><td> Soğutmalı Santrifüj </td><td> Labnet </td><td> 50075040 </td><td></td></tr><tr><td> Nemlendirilmiş İnkübatör CO 2 </td><td> Nuaire </td><td> Model Autoflow 8500 </td><td></td></tr><tr><td> FACSCanto Akış Sitometresi </td><td> BD Biosciences </td><td> 338960 </td><td></td></tr><tr><td> Coulter Partikül Sayacı Z1 </td><td> Beckman-Coulter </td><td> WS-Z1DUALPC </td><td></td></tr><tr><td> Mikrotip ile Sonicator Sıvı İşleme Ekipmanları </td><td> Misonix </td><td> Model No S-4000 </td><td></td></tr></tbody></table>

harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles

Biyobozunur nanopartiküller solunum yolu enfeksiyon hastalıklarına karşı yeni aşıların intranazal tasarlanması ve uygulanması için çok yönlü bir platform olarak ortaya çıkmıştır. Spesifik olarak, polianhidrid alifatik sebasik asit (SA), aromatik 1,6-bis (p-karboksifenoksi) heksan (CPH), ya da amfifilik 1,8-bis (p-karboksifenoksi) -3,6-dioxaoctane oluşan nanoparçacıklar (CPTEG) benzersiz toplu ve yüzey erozyonu kinetik 1,2 gösterilecek ve yavaş yavaş in vivo 3,4,5 fonksiyonel biyomoleküllerin (örneğin, protein antijenleri, immünglobulinler, vb) serbest bırakmak için kullanılabilir. Bu onlara bir aşı dağıtım platformu 6,7,8 için mükemmel bir seçim yapmak, aynı zamanda sahip oldukları içsel adjuvan aktivite nanoparçacıklar. Burun içi mukozal aşılamayı takiben doğuştan gelen bağışıklık aktivasyonu düzenleyen mekanizmaları aydınlatmak üzere, bir antijen p moleküler ve hücre yanıtları değerlendirilmesi gerekirbağışıklık yanıtı başlatmadan sorumlu alınıyoruz hücreler (APC). Alveoler makrofajların (AMɸ) akciğer parankimi 9,10,11 baskın sırasında dendritik hücreler, hava yollarında yürütülmesi bulunan başlıca APC&#39;ler vardır. AMɸ mikrobiyal patojenler ve hücre artıkları 12,13 akciğerlerinde temizlenmesi son derece verimlidir. Ayrıca, bu hücre tipi bir adaptif bağışıklık yanıtının 9 başlamasında önemli bir ilk adımdır lenf düğümleri için mikrobiyal antijenlerin taşıma değerli bir rol oynar. AMɸ da doğuştan örüntü tanıma ve çöpçü reseptörleri, salgılar pro-enflamatuar mediatörler ve başbakan naif T hücrelerinin 12,14 yüksek seviyelerde ifade. AMɸ (ör., daha az% 80) arasında göreceli olarak saf popülasyonunun, laboratuarda çalışma için akciğer lavaj yoluyla elde edilebilir. AMɸ immün yetenekli hayvanlar hasat Resident encounte olacak makrofajların temsilcisi fenotipi sağlamakin vivo r parçacık tabanlı aşı. Bu yazıda, farelerden alınan hasat ve kültür AMɸ için kullanılan protokolleri tanımlamak ve in vitro polianhidrid nanopartiküller ile tedaviyi takiben makrofajların aktivasyonu fenotipi inceleyin.

Watch this Scientific Journal Video about Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles at JoVE.com

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Bu yazıda, akciğer doğuştan gelen bağışıklık hücreleri ikamet ve polianhidrid nanopartiküller ile ko-kültür tepki olarak aktivasyon inceleyerek murin alveoler makrofaj, hasat için protokoller açıklanmaktadır.

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