Bu çalışma Mayo Clinic (CLH) erken bir kariyer geliştirme ödülü, NINDS (CLH) hibe NS64571 tarafından desteklenen ve Donald ve Frances Herdrich (CLH) cömert bir hediye. Biz yardım için Mayo Clinic Sitometrisi Core teşekkür etmek istiyorum.

1. İntrakranial virüs enjeksiyonu: Aşağıdaki teknik değiştirilmiş ve laboratuar ve meslektaşları tarafından yaygın olarak kullanılmıştır. Kısaca, intrakranial enjeksiyon Theiler fare ensefalomiyelit virüsü (TMEV) veya sahte-enfeksiyon Daniel suşu (1, 10) beyin infiltre lökositler (BILs) ortaya çıkarmak için genç farelerde (yaş tercihen 5-6 hafta) yapılır. Daniel zorlanma, fasulye zorlanma ve GDVII suşu arasında farklılık olacağını lütfen unutmayınız. BILs hasat amaçla, fareler TMEV, 2x10 5 PFU almak ve 24 saat için bulaşmış. <ol><li> Otomatik olarak 1 ml Hamilton şırınga, bir iğne (27 gauge ¼ Kendall) takın ve virüsü 10 ul teslim ayarlayabilirsiniz. </li><li> 10 mcL DMEM içinde TMEV hava kabarcıkları dikkat şırıngaya çekin. Uygun olduğunda, sahte enfekte olmuş fareler virüs DMEM 10 ul alırsınız. </li><li> Enjeksiyondan hemen önce, izofluran yaklaşık 1 ml altında pamuk substrat ile bir tel ızgara içeren bir fanus içine dökün. </li><li> Toe-tutam duyarsız ve uçucu anestezik immobilize çan kavanoz tel ızgara üzerine koyun fareler direclty hafif anestezi olana kadar, yaklaşık 10-15 saniye. Fare rahatsız edici ve kostik madde gibi, izofluran ile doğrudan temas etmemelidir. </li><li> Fareler anestezi altında iken, tıraş ve mürekkep kullanılarak kafatası frontal korteks bölgede hızlı bir şekilde uygun koordinatları enjeksiyon için hazırlar. Enjeksiyon için genel konumu bregma için ön 1 mm ve 1 mm sagital sütür (Şekil 1) sağ tarafta lateral. Stereotaktik enjeksiyon bazı deneysel durumlar için uygun olsa da, biz, mürekkep ya da tıraş olmadan ücretsiz el enjeksiyon deneylerin çoğunluğu için uygun olduğunu buldular. Her iki eksen boyunca iğne ucu ile kürk Kesme enjeksiyon için uygun. </li><li> Bir enjeksiyon yapmak için, şark yaklaşık 3 mm derinliğe kadar kafatası ve kemik aracılığı ile basın iğne dik. </li><li> Hamilton şırınga düğmesine basarak ve çekilmeden önce beyin girmek için virüs için 2 saniye bekledikten beyin içine Express virüs. </li><li> Dikkatlice iğneyi çıkarın ve temiz, kuru bir kafes içinde fare. Fare anesteziden hızlı bir şekilde uyanırsınız ve birkaç dakika içinde normal işlevini devam ettirmek. Hayvan ötenazi olarak enjeksiyon yerinde aşırı kanama, anormal belirtileri sergileyen fareler uygun önem vermek uygun olabilir. Uygun hayvan Sağlığı ve Kurumsal Hayvan Bakım ve Kullanım Kurulu kurallar National Institutes 6 ve bağlılığı ele takip edilmelidir. </li></ol> 2. Beyin-infiltre lökosit hücre hazırlama: <ol><li> , 10 ml RPMI 1640, 9 ml Percoll ve 1 ml 10X PBS ve tüpler beyin kaldırma işlemi sırasında, bankta RT oturup, 30 ml kapasiteli tutabilir yuvarlak alt Oak Ridge santrifüj tüplerine hazırlayın. </li><li> Oda sıcaklığında (RT) Ficoll-Paque Plus getirin. </li><li> Değiştirilmiş izofluran doz aşımı 5 hayvan Euthanize ve hızlı bir şekilde buz üzerinde 5 ml RPMI içeren 15 ml konik bir tüp, paslanmaz çelik bir doku spatula ve yer kullanarak beyin kaldırmak. Bazı durumlarda, PBS-perfüzyon BILs hazırlanması periferik kan hücreleri dolaşan kaldırmak için uygun olabilir. Fare, tam ve doğru olarak anestezi öncesinde PBS-perfüzyon olmalıdır. </li><li> Beyin ve 7 ml cam Pyrex marka Tenbroeck doku değirmeni RPMI çözüm aktarın ve hafifçe 10-15 vuruş ile homojenize. </li><li> Homojenizatör ve iki kez pipetlemeyin yukarı ve aşağı doğru pipetleyin ek bir 5 ml RPMI. Transferi beyin Homojenat (yaklaşık 10 ml), önceden hazırlanmış yuvarlak alt tüp ve invert 2-3 kez hafifçe karıştırın. </li><li> Beckman Allegra X-22R klinik santrifüj F0360 sabit açılı rotor RT 30 dakika 7800g ave beyin Homojenat Spin. </li><li> Santrifüj sonra, degrade üst yüzen miyelin enkaz kaldırmak. Kırmızı kan hücresi pelet (Şekil 2) yukarıda yüzer lökosit tabakası toplayın. </li><li> 50 ml konik içine lökosit tabakası, 40 mikron hücre süzgecinden süzün. RMPI ile 50 ml hacmi çözüm getirin. </li><li> Spin tüpler RT az 5 dakika 1500 rpm (600g ave) bir klinik santrifüj içeren seyreltilmiş hücre süspansiyonu. </li><li> Süpernatantı aspire ve hücre pelletini toplamak. 1 ml FACS Tampon pelet (% 1 sığır serum albumin,% 0.02 sodyum kalsiyum azid ve magnezyum PBS) süspanse edin ve 5 ml yuvarlak alt FACS tüpler hücre süspansiyonu aktarmak. </li><li> 1 ml Ficoll-Paque Plus ile dikkatlice altında yatan süspansiyon. </li><li> 2500 rpm (1400 verdi) hiçbir fren RT 25 dakika klinik santrifüj tüp dönerler. </li><li> 1 ml pipet ve yeni 5 ml FACS tüp transfer arabirimi (Şekil 2) beyaz, kabarık katmanı kaldırın. Yıkama c4 ml FACS tamponu ile arşın. </li><li> Pelet hücrelerin klinik santrifüj RT az 5 dakika 1500 rpm (600g ave) tüp dönerler. </li><li> Süpernatant aspire ve hücre pelletini toplamak. Lökosit pelletini tekrar uygun tampon ve yeni bir FACS tüp içinde buz üzerinde tutmak. </li><li> Tripan mavi dışlama hücre canlılığını değerlendirmek ve flow sitometri ile analiz, hücre güvenin. Genel olarak, araştırmacılar yaklaşık 5 x 10 5 hücre / hayvan satın almak için bekleyebilirsiniz. </li></ol> 3. Akış sitometrik işaretleyicidir <ol><li> Lökosit sayımı ve izole sonra, klinik bir santrifüj 1500 rpm (600 verdi), 3 dakika boyunca hücrelerinin dönerler . </li><li> Aşağıdaki içeren tampon engelleme pelletini tekrar: 2.4G2 hibridoma (Fc blok; anti-CD16/32) supernatant FACS tampon, 10:05:01 &#39;lik bir oran fetal sığır serumu ve 4 30 dakika boyunca inkübe ° C . </li><li> Hazırlayın ve 4 1:200 ve inkübe bir konsantrasyon az 30 dakika süreyle bloke hücreleri ° C karanlıkta konjuge ekstrasellüler antijenlere karşı antikorlar ekleyin. Leke ve 200 ul hücreler, bir 96-iyi V-alt plaka kuluçkaya yatmaktadır. Uygun kontroller gerekli lekesiz hücreleri ve florokrom tazminat örnekleri, içerir. </li><li> 96-kuyucuğu rotor uygun bir klinik santrifüj 1500 rpm (600 verdi) 3 dakika RT boyanan hücrelerin dönerler. </li><li> Süpernatantı aspire ve hücreleri hafifçe aşağı yukarı 3 kez pipetleme ve 200 ul FACS tampon ile yıkayın. </li><li> Yukarıdaki yıkama tekniği iki kez tekrarlayın. </li><li> Yıkandıktan sonra,% 2 paraformaldehid FACS tüplerine transfer hücreleri tekrar süspansiyon haline getirin. Fixation Biyogüvenlik Seviye 2 reaktiflerin enfekte hücreleri akım sitometri analizi için gereklidir. </li><li> Değiştirilmiş bir akım sitometri yöntemi 8 BD FACS Calibur sabit hücreler çalıştırın ve çevrimdışı FlowJo, WinMDI veya diğer mevcut flow sitometri analiz yazılımı kullanarak dosyaları analiz. </li><li> Örnek başına 50-100,000 olayların analizi genellikle yeterli işaretleyicidir için gereklidir. </li></ol> Bu deneyde kullanılan doğrudan konjuge antikorları: CD45 klon 30-F11 ile tespit edildi. Ly6C / G klon Gr1, RB6-8C5 ile tespit edildi. CD11b klonu M1/70 ile tespit edildi. F4/80 klonu BM8 ile tespit edildi. 4. Temsilcisi Sonuçlar: Biz 24 saat sonrası enfeksiyon (Şekil 3) fare BILs için hücre fenotipleme sonuçları göstermektedir. Lökositler, bağışıklık hücreleri algılamak için PerCP-konjuge anti-fare CD45 ile boyandı, PE-konjuge anti-fare Ly6C / G hücreleri algılamak için inflamatuar monositler, APC-konjuge anti-fare CD11b ve APC-konjuge anti-fare F4/80 tespit etmek için monosit soy. BD FACS Calibur enstrüman analizi yapıldı. <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="Şekil 1"/> Şekil 1. Enjeksiyon yerinde Anatomik lokalizasyon. Enjeksiyon yerinde bregma için ön 1 mm ve sağ tarafta sagital sütür 1 mm lateralinde yer alır. <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="Şekil 2"/> Şekil 2. Lökositler ve BILs degrade ayrılması İllüstrasyon lökositler başlangıçta doğrudan miyelin enkaz katmanı altında ve Percoll degrade RBC pelet üzerinde toplanır. Ficoll degrade arayüzünde beyaz, kabarık bir katman (BILs) toplanır. <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="Şekil 3"/> Şekil 3. 24 saat sonrası enfeksiyon Fare lökositlerin beyin infilatrating lökositlerin immünfenotip beyin doku intrakranial enfeksiyon sonrası 24 saat hayvanlar hazırlanan homojenatlarında ayırıcı yoğunluğu santrifüj tarafından izole edilmiştir. Hücreler fare CD45 karşı floresan-konjuge antikor ile boyandı, Ly6C / G, CD11b ve F4/80. İlk yolluk forward scatter (FSC), hücre boyutu bir göstergesi (C) karşı CD45, bağışıklık hücreleri için bir belirteç, komplo yapıldı. CD45 hi (A, D), CD45 lo (B, E), ve CD45 negatif (F, G) popülasyonları ardından monosit ve makrofaj belirteçlerinin Ly6C / G, F4/80 (A, B, göreceli ekspresyon düzeyleri incelendi F) ve CD11b (D, E, G). Ly6C / G + F4/80 + CD11b + inflamatuar monositler çevreden bu hücrelerin infiltrasyonu ile tutarlı CD45 yüksek nüfus (A, D), neredeyse sadece bulundu. Buna karşılık, Ly6C/G-F4/80 lo CD11b + makrofajlar CD45 lo (B, E) ve CD45 negatif (G), yerleşik fenotip ile tutarlı nüfus, neredeyse sadece bulundu . Sayısız deneylerde, CD45 hi bulaşmamış veya sahte-enfeksiyon beyin hücreleri veya Ly6C / G + F4/80 + CD11b + inflamatuar monositler asla riayet varTed fareler (veriler gösterilmemiştir).

<ol>
	<li>Buenz, E. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornaviruses+and+cell+death.">Picornaviruses and cell death.</a> Trends Microbiol. 14, 28-36 (2006).</li><li>Buenz, E. J., Limberg, P. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+3-parameter+flow+cytometry-based+cell+death+assay.">A high-throughput 3-parameter flow cytometry-based cell death assay.</a> Cytometry A. 71, 170-173 (2007).</li><li>Deb, C., Lafrance-Corey, R. G., Zoecklein, L., Papke, L., Rodriguez, M., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelinated+axons+and+motor+function+are+protected+by+genetic+deletion+of+perforin+in+a+mouse+model+of+multiple+sclerosis.">Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.</a> J. Neuropath. Exp. Neurol. 68, 1037-1048 (2009).</li><li>Deb, C., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+contributes+to+efficient+clearance+of+picornavirus+from+the+acutely+infected+murine+brain.">NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain.</a> J. Neurovirol. 14, 261-266 (2008).</li><li>Donovan, J., &amp;amp, P. . B. r. o. w. n., , P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols in Neuroscience. , A.4H.1-A.4H.4 (2005).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+&#38;+restraint.">Handling &#38; restraint.</a> Current Protocols in Immunology. 73, 1.3.1-1.3.6 (2006).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Current Protocols in Neuroscience. , A.4F.1-A.4F.9 (2005).</li><li>Holmes, K. L., Otten, G., Yokoyama, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+using+Becton+Dickinson+FACS+Calibur.">Flow cytometry analysis using Becton Dickinson FACS Calibur.</a> Current Protocols in Immunology. , 5.4.1-5.4.22 (2002).</li><li>Howe, C. L., Ure, D., Adelson, J. D., LaFrance-Corey, R., Johnson, A., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+directed+against+a+viral+peptide+contribute+to+loss+of+motor+function+by+disrupting+axonal+transport+in+a+viral+model+of+fulminant+demyelination.">CD8+ T cells directed against a viral peptide contribute to loss of motor function by disrupting axonal transport in a viral model of fulminant demyelination.</a> J Neuroimmunol. 188, 13-21 (2007).</li><li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+An+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: An unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li></ol>

Biz rutin beyin infiltre hücre hazırlama kalitesini belirlemek için flow sitometri ve bağışıklık hücrelerini 2, 9 farklı nüfusu ayırt etmek. Akut zaman noktalarında, BILs yöntemi CD45hi CD45lo nüfus makrofajların yanı sıra yüksek oranda nüfus içinde yüksek oranda inflamatuar monositlerin verir. Bu, beynin içinde bir tekrarlanabilir immün yanıt sağlam yöntemi ile karakterize edilebilir olduğunu gösterir. Bu tekniği fare beyin bağışıklık hücreleri...

<table border="1"><tbody><tr><th> Reaktif </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> TMEV </td><td> Howe Lab. </td><td> N / A </td><td></td></tr><tr><td> Daniel&#39;in en gerginlik </td><td> Invitrogen </td><td> 21063-029 </td><td></td></tr><tr><td> izofluran </td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td> yuvarlak alt Oak Ridge santrifüj tüplerine </td><td> Nalgene </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-02 </td><td></td></tr><tr><td> 10X PBS </td><td> Roche </td><td> 11666789001 </td><td></td></tr><tr><td> Ficoll-Paque Plus </td><td> GE Healthcare </td><td> 17-1440-02 </td><td></td></tr><tr><td> Tripan Mavi 0.4% (w / v) </td><td> Mediatech </td><td> 25-900-CI </td><td></td></tr><tr><td> 15 ml konik tüpler </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> 7 ml&#39;lik cam Pyrex marka Tenbroeck doku değirmeni </td><td> Balıkçı </td><td> 08-414-10B </td><td></td></tr><tr><td> 40 mikron hücre süzgecinden </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 50 ml konik </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> sığır serum albumini </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> sodyum azid </td><td> Sigma </td><td> S8032 </td><td></td></tr><tr><td> CMF-PBS </td><td> Mediatech </td><td> 21-040-CV </td><td></td></tr><tr><td> FACS borular </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> fetal sığır serumu </td><td> Sigma </td><td> F4135 </td><td></td></tr><tr><td> 2.4G2 hibridoma </td><td> ATCC </td><td> HB-197 </td><td></td></tr><tr><td> Costar V-alt plaka </td><td> Corning </td><td> 3894 </td><td></td></tr><tr><td> Allegra X-22R santrifüj veya eşdeğeri </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> 96-plaka kova ve rotor </td><td> Beckman </td><td> S2096 </td><td></td></tr><tr><td> Sabit açılı rotor </td><td> Beckman </td><td> F0360 </td><td></td></tr><tr><td> paraformaldehid </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> BD FACS Calibur </td><td> BD Biosciences </td><td> N / A </td><td></td></tr><tr><td> FlowJo 7.5 </td><td> Tree Star, Inc. </td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD Biosciences </td><td> 557235 </td><td> klon: 30-F11 </td></tr><tr><td> Gr1 (Ly6C / G) </td><td> BD Biosciences </td><td> 553128 </td><td> klon: RB6-8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td> klon: M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td> klon: BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

Biz farelerin beyin infiltre lökositler (BILs) hazırlamak için bir metodu tanımlar. Biz tüm beyin hücrelerinin infiltre bir lökosit zenginleştirilmiş nüfus nasıl arındırmak için hızlı bir intrakraniyal enjeksiyon tekniği ile Theiler fare ensefalomiyelit virüsü (TMEV) ile enfekte fareler nasıl gösterilmektedir. Kısaca, fare, kapalı bir odasında izofluran ile anestezi ve serbest el frontal korteksin içine Hamilton şırınga ile enjekte edilir. Fare sonra bir Tenbroeck doku değirmeni ile RPMI ayıklanır ve homojenize izofluran doz aşımı ve bütün beyinleri tarafından enfeksiyondan sonra değişik zamanlarda öldürüldü. Beyin homojenatlarında miyelin ve diğer hücre enkaz kaldırmak için sürekli% 30 Percoll degrade ile santrifüj edilir. Hücre süspansiyonu daha sonra 40 mikron gergin, yıkanmış ve süreksiz Ficoll-Paque Plus degrade santrifüj lökosit seçin ve arındırmak için. Lökosit sonra yıkanır ve flow sitometri ile işaretleyicidir için uygun tamponlar yeniden süspanse. Flow sitometri, enfeksiyonun erken aşamalarında C57BL / 6 farelerde doğuştan gelen bağışıklık hücreleri bir nüfusa ortaya koymaktadır. 24 saat enfeksiyonu, bağışıklık hücrelerinin birden fazla altkümelerini Gr1 +, CD11b + ve F4/80 + hücreler zenginleştirilmiş bir nüfusa sahip, BILs mevcut . Bu nedenle, bu yöntem, beyinde akut enfeksiyon bağışıklık yanıtı karakterize yararlıdır.

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

Fare beyni infiltre lökositler elde etmek için hızlı bir yöntem açıklanmıştır. Bu yöntem, sürekli Percoll degrade ve süreksiz Ficoll degrade lökosit zenginleştirilmiş katmanı seçin ve arındırmak için kullanır. İzole lökositler akım sitometrik ölçümleri ile karakterize olabilir.

Beyin-infiltre lökositler izolasyonu

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

isolation-of-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K Görüntüleme.  Mayo Clinic College of Medicine. Fare beyni infiltre lökositler elde etmek için hızlı bir yöntem açıklanmıştır. Bu yöntem, sürekli Percoll degrade ve süreksiz Ficoll degrade lökosit zenginleştirilmiş katmanı seçin ve arındırmak için kullanır. İzole lökositler akım sitometrik ölçümleri ile karakterize olabilir.

Video: Isolation of Brain-infiltrating Leukocytes

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.

Fonksiyonel Kardiyak Bağışıklık Hücreleri İzolasyon

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 ...

İmmünoloji ve Enfeksiyon

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Fare Akciğer Dendritik Hücrelerinin İzolasyonu

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Den Beyin sekestre Lökositler İzolasyonu ve Analizi<em&gt; Plasmodium berghei</em&gt; ANKA-enfekte Fare

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

İnsan lökosit ve trombosit İzolasyonu ve Değerlendirme Protokolleri: Biyoenerji ve Oksidatif Burst

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Akış adlı lökositlerin üzerine Stromal hücreler Etkilerinin İncelenmesi

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

İnsan Maternal-Fetal Arabirimi lökosit İzolasyonu

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Maternal Fetal Arayüz at Fare Dokular lökosit İzolasyonu

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Enzimatik Digest ve Gradient Ayırma kullanma Fare Skin gelen sızan lökositlerin İzolasyonu

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating pulmoner lökosit seçici Hasat

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatik lökosit seçici Hasat

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...