Biz biyogüvenlik düzeyi 2 örneklerinden birincil fare AECII sıralama içinde teknik yardım için M. Höxter teşekkür etmek istiyorum. Bu çalışma DB Alman Araştırma Topluluğu (DFG) (SFB587, TP B12 ve BR2221/1-1) ve Hannover Biyomedikal Araştırma Okulu (DFG GSC 108) AA bir nakit hibe ile desteklenmiştir. DB Başkan Girişimi ve sözleşme numarası W2/W3-029 altında Alman Araştırma Merkezleri Helmholtz Derneği (HGF) ve Ağ Fonu tarafından desteklenmektedir.

Gerekli reaktifleri ve materyalleri ilişkin ayrıntıları aşağıda protokolünün sonunda tabloda listelenmiştir. Çalışmaya başlamadan önce, 4 dispase ml ve ° C su banyosunda 37 öncesi ılık bunları içeren 15 ml tüpler (fare başına bir) hazırlamak. Bir ısıtma bloğu, kısaca 95-1% düşük erime agaroz (su) küçük alikotları ısıtmak ° C sıvılaştırılmış kadar ve kullanıma kadar 45 ° C ye sonradan serin. 1. Fare Akciğer hazırlanması <ol><li> CO 2 boğulma tarafından fare Kurban. Not: başarıyla yapılamaz, böyle bir sıvı ile akciğer kurulum gibi, protokol adımları izleyerek, böylece bu trakea yaralama gibi servikal dislokasyon yapmayın. Nosiseptif reflekslerinin kaybı olun. </li><li> Etanol ile fare Sprey ve vücut ventral orta hat boyunca uzun bir kesim yapmak. Ventral kürk ve deri çekin ve daha sonra da dikkatlice kesilmiş ve periton çıkarın. </li><li> ExsanguinateSağ ve sol juguler ven (laktasyonun) yanı sıra renal arter (Arteria renalis) keserek fare. Doku ile akan kan çıkarın. </li><li> Dikkatlice sonra delinme ve kaburga keserek kalp ve akciğer maruz sökmek. Akciğer kesmemeye özel dikkat gösterin. </li><li> Soğuk fosfat dolu bir 26G kanül ve 10 ml şırınga ile salin (PBS), ponksiyon kalbin sağ ventrikül tamponlu ve kan serbest kadar PBS ile akciğer serpmek. </li><li> Trakea maruz tükürük bezlerinin kesin ve çıkarın. Ayrıca dikkatle trakea çevreleyen kas kesti. </li><li> Trakea içine 22G kanüllerin yerleştirin, iğneyi çıkarın ve akciğer doğru plastik kateter itin. Trakea ve kateter etrafında iplik küçük bir parça bağlayarak kateter Fix. </li></ol> 2. Akciğer Doku Enzimatik Sindirim İsterseniz, bronkoalveoler lavaj sıvısı örneği olabiliradımdan önce PBS veya orta trakea takılı kateter yoluyla akciğerlere kızarma tarafından hazırlanmıştır. <ol><li> 2 ml&#39;lik şırınga yardımıyla, her lobun tam olarak genişletilmiş, böylece kateter yoluyla akciğer içine dispase 2 ml (4 ml bir örnek gelen) bir maksimum hacim aşılamak. Sıvılaştırılmış agaroz 0.5 ml içeren 1 ml şırınga ile şırınga değişim ve aynı zamanda akciğer içine aşılamak. </li><li> Agaroz geri akışını önlemek ve hemen laboratuar kağıt mendil ve buz ile akciğer karşılamak için kateter üzerinde şırınga bırak. Birkaç dakika için agaroz jel olsun. </li><li> , Kağıt mendil, buz, şırınga ve kateter çıkarın trakea ve tüketim akciğer, kalp ve göğüs timus kesti. Sonra PBS ile bir tabak içinde akciğer durulayın ve kalp, timus ve kalan trakea çıkarın. </li><li> Dispase geri kalan kısmı 2 ml akciğer içine yerleştirin ve 45 dakika süre ile oda sıcaklığında inkübe edilir. </li></ol> 3. Akciğer hazırlanmasıHücre Süspansiyon <ol><li> Dispase gelen akciğer çıkarın ve DMEM ortamı ve 100 ul içinde DNase anti-CD16/32 antikor 7 ml (1 ug / ml nihai konsantrasyon) ihtiva eden bir çanak içine aktarın. </li><li> Forseps kullanarak, tamamen ayrı çekerek akciğer doku parçalanır ve yavaşça 200 rpm ayarlanmış bir rocker üzerinde sallanan ise oda sıcaklığında 10 dakika inkübe edilir. Arzu edildiği takdirde, hücre süspansiyonu daha sonra bir sonraki adıma kadar 4 ° C de muhafaza edilebilir. </li><li> 100 mikron ve daha sonra 70 mikron, 48 mikron ve 30 mikron gözenek ile ilk naylon kafesleri yoluyla hücre süspansiyonu filtre. Hücre kaybını en aza indirmek ve verimi en üst düzeye çıkarmak için de DMEM ile tüpler ve iyice yemekleri gibi her örgü durulama özen gösterin. Eğer örnek birleştirilmesi, bu daha sonra aynı filtre ile bir grup farelerden alınan hücreler süspansiyonlar geçerek burada elde edilebilir. Tıkanma durumunda filtre yenileyin. </li><li> Hacmine bağlı olarak, bir tane ya da daha fazla 50 mL tüpler ve süzüntü aktarmak160 x g &#39;de 15 dakika, 4 ° C&#39;de santrifüj Süpernatantı. </li><li> 2 ml eritrosit lizis tamponu (0.15 M NH4CI, 0.01 M KHCO 3, 0.1 mM EDTA, pH 7.2) içinde yeniden süspanse edin ve hücre peleti hızla DMEM ortamı 13 ml ilavesiyle lizis sona erer. 160 x g&#39;da 12 dk, 4 ° C&#39;de bir 15 ml tüp ve santrifüj çözelti aktarın </li></ol> 4. Akış Sitometrik Hücre-sıralama için Antikor Boyama <ol><li> Akış sitometrisi için uygun dilüsyonlarda DMEM ortamı içinde hazırlanmış, 3 ml birincil antikor kokteyli, hücrelerin yeniden süspanse edin. (Antikorlar, önceden 100 ul bir hacim içinde 1 x 10 6 splenositlerin lekeleme ile optimum çalışma dilüsyonları için titre edilmiştir. Kullanımlar beş fare kadar birikintilerini hücreler için kullanılan antikorların her biri için optimum konsantrasyonu ihtiva eden birincil antikor kokteyli 3 ml. Daha fazla fareler bir örneğe toplanmış ise,) seviyesini ayarlamak. </li><li> Boyanması için, karanlıkta 10 dakika için hücreler inkübe4 ° C&#39;de </li><li> 12 160 x g&#39;de ve 4 dakika için santrifüjleme ile DMEM ortam ile 15 ml tüp doldurma ile yıkayın hücrelerini ° C. </li><li> Halinde ya da bağlanmamış biotinlenmiş antikorları 4.1 kullanılmıştır: süpernatantı ve 2 hücreleri tekrar süspansiyon - 3 ml ikincil antikor kokteyli. Karanlıkta 4, 10 dakika ° C&#39;de Leke. </li><li> 160 x g&#39;da 12 dk, 4 ° C ve DMEM ve santrifüjleme ile tüp doldurma yoluyla yıkayın hücrelerini </li><li> Hücre-sıralama için bir tüp içine bir 50 um filtre aracılığıyla DMEM ve ön-filtre, 1 ml hücre yeniden süspanse edin. İsteğe bağlı: akciğer hücre süspansiyonu toplam hücre sayısını elde etmek için hücreleri saymak. </li></ol> 5. Hücre-sıralama <ol><li> Sıralama önce vorteks hücreleri karıştırın. AECII hücre-sıralama için 100 mikron nozul kullanın. Kılıf sıvı basıncı hem de lazer emisyon ve saptama dalga boyları akış sitometrik hücre-sıralama gibi antib kullanılan florokromlar için kullanılan cihaza bağlıdırody boyama işlemi. </li><li> DMEM içeren bir tüpe boyama ve sıralama için kullanılan florokromlar için negatif SSC Yüksek hücreleri üzerinde Kapısı. (SSC-A) pencereler (içinde yolluk stratejisinin ayrıntılarını gördüğünüz herhangi çiftler veya hücre agrega dışlamak için dağılım yüksekliği (FSC-H) vs alanı (FSC-A) ve yan dağılım yüksekliği (SSC-H) vs alanında ileri kullanın 1) Şekil. </li><li> 20 280 xg&#39;de dk ve 4 kriteri hücreler santrifüj toplamak amacıyla ° C. </li><li> Daha sonraki analiz için gerekli olan kültür ortamı ya da tampon olarak hücreler yeniden süspanse edin. </li></ol>

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Çıkar çatışması ilan etti.

Flow sitometri ile murin AECII ve izolasyonu için bizim protokol fonksiyonel ve moleküler çalışmalar bir dizi için fare akciğer primer hücrelerin erişmenin hızlı bir yol sunar. Açıklanan prosedür böyle bir RNA izolasyonu (şekil 2b bakın) ve transcriptome çalışmaları gibi doğrudan sonraki analizler için sayıca yeterli AECII son derece canlı ve saf popülasyonlarının verir. Fonksiyonel uygulamalar için, AECII şartlandırılmış ortam ya da ko-kültür deneylerinden elde edile...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> kanüllerin Introcan 22G </td><td> Braun </td><td> REF 4252098B </td><td></td></tr><tr><td> Dispase, 100 ml (5000 caseinolytic birimleri) </td><td> BD Biosciences </td><td> 354235 </td><td> 15 ml tüp içinde 4 ml kısım, -20 ° C de saklamak </td></tr><tr><td> Biozym Plak Agaroz </td><td> Biozym </td><td> 840101 </td><td> H2O içinde% 1 w / v </td></tr><tr><td> Sığır pankreas, 2000 Kunitz birimleri / şişeden deoksiribonükleaz Ben </td><td> Sigma-Aldrich </td><td> D4263 </td><td> taze 300 ul DMEM 1 flakon içeriği çözülür </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 22320-022 </td><td> kullanılan üreticisi (Low G tarafından sağlananlucose, Piruvat, HEPES) </td></tr><tr><td> hücre süzgeçler (100 mikron, 75 mikron) </td><td> BD Falcon </td><td> 352.360, 352.350 </td><td></td></tr><tr><td> naylon elekten (48 um, 30 um) </td><td> Buckmann GmbH </td><td> 03-48/26-1020, 03-30/18-108 </td><td></td></tr><tr><td> CellTrics 50 um bir filtreden </td><td> Partec </td><td> 04-0042-2317 </td><td></td></tr><tr><td> anti-fare CD16/CD32 </td><td> BioLegend </td><td> 101302 </td><td> klon 93; arıtılmış </td></tr><tr><td> anti-fare F4/80 </td><td> BioLegend </td><td> 123116 </td><td> klon BM8; APC kuplajlı </td></tr><tr><td> anti-fare CD11b </td><td> BioLegend </td><td> 101208 </td><td> klon M1/70; PE kuplajlı </td></tr><tr><td> Anti-CD11c fare </td><td> BioLegend </td><td> 117310 </td><td> klon N418; APC kuplajlı </td></tr><tr><td> anti-fareCD45 </td><td> BioLegend </td><td> 103102 </td><td> klon 30-F11; saflaştırılmış </td></tr><tr><td> anti-fare CD19 </td><td> eBioscience </td><td> 12-0193-83 </td><td> eBio 1D3; PE kuplajlı </td></tr><tr><td> poliklonal keçi anti-fare IgG </td><td> BD Pharmingen </td><td> 550767 </td><td> poliklonal, PE kuplajlı </td></tr><tr><td></td><td></td><td></td><td> Alternatif florokromlar ile birleştirilen antikorlar mevcut akış sitometresi ve lazer bağlı olarak kullanılabilir. </td></tr></tbody></table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

Geçen yıllar boyunca, akciğer immün regülasyon çeşitli yönleriyle alveolar tip II epitel hücreleri (AECII) katkısı giderek kabul edilmiştir. AECII iltihaplı hava yollarında sitokin üretimini katılmak ve enfeksiyon ve T-hücre aracılı otoimmünite 1-8 hem de hücreler antijen sunan bile hareket etmek gösterilmiştir. Bu nedenle, bu tür hava yolu hiper reaktivitesi gibi klinik bağlamlarda da özellikle ilginç yabancı ve hem de self-antijenlere doğrudan veya dolaylı AECII hedef enfeksiyonlar. Bununla birlikte, sağlıklı akciğer yanı sıra enflamasyon alveolar tip II epitel hücreleri tarafından sunulan ayrıntılı immünolojik işlevlerini anlaşılmasında düzensizdir. AECII fonksiyonu ile ilgili birçok çalışma 9-12 fare, veya insan alveoler epitel hücre hatları kullanılarak yapılmaktadır. Hücre hatları ile çalışmak kesinlikle böyle büyük sayılar durumu o gibi, faydaları sunarkapsamlı analizler için f hücreler. Bununla birlikte, birincil murin AECII kullanımı ile enfeksiyon ya da otoimmün iltihabı gibi karmaşık işlemler olarak bu hücre tipinin rolü daha iyi anlaşılmasını sağlar inanıyoruz. Primer mürin AECII bunlar analiz ortamında bir rol oynayan ek dış faktörler işlemine tabi tutulmuştur, yani bu gibi solunum durumları olan hayvanlardan doğrudan izole edilebilir. Bir örnek olarak, uygun intranazal AECII öncelikle çoğaltma 13 için bu hücreler hedef grip A virüsü ile enfekte olan fareler izole edilebilir. Önemlisi, sağlıklı farelerin izole AECII ex vivo enfeksiyon yoluyla, enfeksiyon üzerine monte hücresel cevaplarının çalışmalar daha uzun olabilir. Birincil mürin AECII ve izolasyon için protokol, CD11c, CD11b, F4/80 için spesifik antikorlar ile elde edilen hücre süspansiyonu etiketleme ardından fare akciğer enzimatik sindirim dayanmaktadırCD19, CD45 ve CD16/CD32. Granül AECII sonra yüksek etiketsiz ve yan dağılım (SSC yüksek) hücre popülasyonu olarak belirlenmiş ve 3 sıralama floresan aktive hücre ile ayrılır. Fare akciğerlerinden primer epitelyal hücreler izole alternatif yöntemler karşılaştırıldığında, negatif seçimin AECII akım sitometri izolasyonu için bizim protokol nispeten kısa bir süre içinde dokunulmaz, son derece canlı ve saf AECII verir. Buna ek olarak, ve kaydırma ve lenfosit azalması ile izolasyon geleneksel yöntemlerin aksine, antikor-coupled manyetik boncuklar 14, 15 bağlayıcı aracılığı ile, akış sitometrik hücre-hücre boyut ayırma ve ayrıntı ile ayırt etme izin verir. Akım sitometrik hücre sıralama için enstrümantasyon kullanılabilir olduğunu göz önüne alındığında, açıklanan prosedür nispeten düşük maliyetle uygulanabilir. Standart antikor ve akciğer dağılma için enzimler, manyetik olarak herhangi bir reaktif madde yanındaboncuklar gereklidir. İzole hücrelerinin in vitro kültürü ve T-hücre uyarma deneylerinde de transcriptome, proteom veya secretome analizi 3, 4, olarak dahil işlevsel ve klinik çalışmalar, geniş bir aralığı için uygundur.

Sağlıklı farelerden izole akciğer hücre süspansiyonları sıralama yapılırken AECII kapısı genellikle yaklaşık 42 hesap verecek ± tüm olayların 10%. Bu yüzde, başlangıçtaki hücre süspansiyonu solunum yolları için işe lenfositler ve diğer bağışıklık hücrelerinin oldukça yüksek oranda içerecek şekilde, örneğin bir viral enfeksiyon gibi solunum koşullar ile farelerin kullanılması durumunda belirgin ölçüde daha düşük olabilir. Günde 3 Aşağıdaki enfeksiyonu IAV enfekte akciğ...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

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

Biz flow sitometrik negatif seçimin birincil fare tip II alveol epitel hücrelerinde (AECII) hızlı izolasyonu tarif. Bu AECII yüksek canlılığı ve saflık göstermektedir ve bu gibi otoimmün veya bulaşıcı hastalıklar gibi solunum koşullar altında rolü ile ilgili işlevsel ve klinik çalışmalar, geniş bir aralığı için uygundur.

Fonksiyonel ve Moleküler Çalışmalar Birincil murin Tip II alveol epitel hücrelerinde akım sitometri İzolasyonu

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

flow-cytometric-isolation-primary-murine-type-ii-alveolar-epithelial

Research

JoVE Journal

Immunology and Infection

26.1K Görüntüleme.  Helmholtz Centre for Infection Research. Biz flow sitometrik negatif seçimin birincil fare tip II alveol epitel hücrelerinde (AECII) hızlı izolasyonu tarif. Bu AECII yüksek canlılığı ve saflık göstermektedir ve bu gibi otoimmün veya bulaşıcı hastalıklar gibi solunum koşullar altında rolü ile ilgili işlevsel ve klinik çalışmalar, geniş bir aralığı için uygundur.

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

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.

Bağışıklık Hücreleri murin aorta içinde Sitometrisi Analizi

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

İmmünoloji ve Enfeksiyon

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Yapımcı Epitel Hasar Analizi<em&gt; Entamoeba histolytica</em&gt; Enfeksiyon

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

İskemik Fare Beyin gelen Bağışıklık Hücreleri İzolasyon ve Akım Sitometrisi Analizi

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

İzolasyon ve Glioma-sızmakta periferik kan mononükleer hücrelerinin Akışı Sitometrik Analizi

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Fare Küçük Bağırsak Lenfositler izolasyonu ve Akım Sitometrisi Karakterizasyonu

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

İnsan Endoservikal Gamma Delta T Hücreleri İzolasyon ve Akım Sitometrisi Analizi

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Lenfatik endotel hücreleri akış sitometrik çözümlenmesi için fare karaciğer sindirim

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...