Bu çalışma, bir araştırma hibe ve Hibe Yardım, Japonya Tarım, Orman ve Balıkçılık Bakanlığı Gıda Nanoteknoloji Projesi tarafından finanse edildi

1. Karaciğer, Perfüzyon <ol><li> Intraperitoneal sodyum pentobarbital bir çözüm (50 mg sodyum pentobarbital / kg vücut ağırlığı); yetişkin erkek Sprague-Dawley sıçan (vücut ağırlığı 150-200 gr hafta-10-15) anestezisi. </li><li> Paslanmaz bir tavada hayvan koyun ve% 70 etanol, karın ve göğüs üzerine püskürtün. </li><li> Açıklarındaki küçük bir kesim ve cilt soyma olun. Bir makas ile kesme karın açın. </li><li> Portal ven yerini onaylayın, portal ven ve gevşek bir tutam altında bir cerrahi iplik geçer. </li><li> Tutam inferior vena kava ve karaciğerde kanın geri kaçmasını önleyecek şekilde mezenterik ven distal parçası. Göğüs boşluğu diyafram makasla keserek açın ve kalp maruz. </li><li> Plastik bir kateter (2 mm çapında) ve portal ven içine yerleştirin ve kateter etrafında konu iyice sıkın. </li><li> 37 ° prewarmed ° C; yıkama solüsyonu (pH 7,2 Ca 2 +-Mg 2 + 0,5 mM EGTA, 10 mM HEPES ve 4.2 mM NaHCO 3 içeren ücretsiz HBSS) ile yüklenen bir perfüzyon sistemi kateter bağlayın </li><li> 10 ml / dak hızında in situ perfüzyon başlayın. Aynı zamanda, sağ atrium duvarında bir kesim olun. 10 dakika için perfüzyon devam edin. </li><li> Perfüzyon çözüm kollajenaz çözüm Anahtarı [Tipi ile desteklenmiş 10mM Hepes ve 4.2mm NaHCO 3 içeren HBSS kollajenaz (% 0.05) ve tripsin inhibitörü (50 mg / ml), pH 7.5] 10 ve serpmek hızında 20 dakika 10 ml / dak karaciğer biraz şişer ve seröz membran altında karaciğer yapısının dağılması için işareti gösterene kadar. </li></ol> 2. Parankimal Hepatosit zengin Hücre Kesir hazırlanması <ol><li> Çıkarın ve dikkatli bir şekilde steril bir behere 25 ml kollajenaz çözüm içeren karaciğer transferi. Makasla küçük parçalar halinde Kıyma. </li><li> Ortaya çıkan hücre süspansiyonu içine 75 ml soğuk MEM ekleyin. Nazik pipetleme sonra, süzüntü bir hücre süzgeç (100 mm) ile, bağ dokuları ve sindirilememiş doku parçaları kaldırmak için. </li><li> 4 ° C (fren kapalı) 1 dakika 50 xg&#39;de süzüntü 50 ml konik tüpler ve santrifüj içine aktarın. </li><li> Süpernatantı dikkatli atın. Soğuk MEM pelletini tekrar. </li><li> ) Üç kez 2.4 tekrarlayın. </li></ol> 3. Karaciğer Hücreleri Karma İlköğretim Kültür <ol><li> DMEM 100 mcM β-mercaptoethanol 10 mg / ml insülin, 100 mg / ml streptomisin ve 100 U / ml penisilin ve tohum ile desteklenmiş% 10 ısı inaktive FCS içeren oluşan besi 2.5 &#39;te elde edilen hücreler) Askıya Alma 6.7 x 10 4 hücre / cm 2 (orta 5 x 10 6 hücre / şişeyi / 12 -15 ml) yoğunluğu: 09:55 doku kültürü şişeleri (75 cm 2 yüzey alanı). </li><li> Inkübe kültür flasksat 37 ° C -% 5 CO 2 &#39;nin bir atmosfer% 95 hava. </li><li> Besi her 2 ila 3 gün aralıklarla değiştirin. </li></ol> 4. Makrofajlar, Seçici İzolasyon <ol><li> 12 gün sonra kültür, makrofaj hücre kağıda çoğalırlar. Bu hücreler, 80 kültür şişe sallayarak karşılıklı tarafından askıya alınır - 30 dakika için dakikada 120 vuruş 37 ° C </li><li> 100 mm olmayan doku kültürü dereceli plastik tabaklar kültür ortamı içine aktarın. Bir plastik tabak içine iki T75 şişeler orta Havuzu. Büyüme, orta ve Yedek kültür şişeler inkübatör onları geri. </li><li> 37 30 dakika inkübe plastik tabaklar ° C -% 5 CO2 atmosferi% 95 hava Makrofajlar kolayca diğer kirliliğe neden olan hücreleri yok ise, bu kuluçka süreci kapsamında olmayan doku kültürü notu plastik tabaklar eklemek. </li><li> Aspire orta ve PBS ile plastik yemek nazikçe yıkayın. Bu adımı üç kez tekrarlayın. </li><li> TrypLE Express çözümü 1 ml çanak içine ekleyin ve 37 o 10 dakika süreyle inkübe ° C -% 5 CO2 atmosferi% 95 hava. </li><li> Besi 9 ml ekleyin. Bir hücre kazıyıcı bağlı makrofaj hafifçe kazıyın. </li><li> Hücre süspansiyonu, ° C (fren) 5 dakika boyunca 25 180 xg 15 ml konik tüp ve santrifüj içine aktarın. </li><li> Süpernatantı atın ve besi 1 ml ekleyin. Dinç pipetleme tek hücre içine ayrıştırmaları. Hemasitometre sayma hücre sayısı numaralandırılamıyor. </li><li> İzolasyon adımları, 2-3 gün aralıklarla en fazla iki hafta boyunca aynı kültür şişeler tekrarlanmalıdır. </li><li> İzole makrofajlar 3.1 &#39;de açıklanan büyüme ortamında yetiştirilebilir. </li></ol> 5. Temsilcisi Sonuçlar Yetişkin sıçan karaciğer hücrelerinin karışık bir birincil kültürünün bir örneği Şekil 2&#39;de gösterilmiştir. Parankimal hepatositler kültür birkaç gün içinde epitel hücre morfolojisi kaybedersiniz, yozlaşmış ya da fibroblast-benzeri hücreler haline dönüştürmek.Sonra, günde yaklaşık 6 ila 12, faz kontrast parlak, yuvarlak makrofaj benzeri hücreler vigourouly fibroblastik hücre kağıda çoğalırlar. Kültür şişe çalkalanarak, makrofajlar kültür ortama kolayca askıya alınır ve plastik tabaklar sonraki transfer ve inkübasyon makrofaj seçici yapışma (Şekil 3). Makrofaj-benzeri hücreler 8. günde olduğu kadar erken olarak hasat ve sayıları 12 ila 14 gün maksimum seviyelere ulaşmıştır. Fazla 10 6 hücre kültür şişesi (Şekil 3) 10 7 T75 her bir şişesi ortalama toplam hücre verimi sağlayan, en fazla iki hafta boyunca 2 ila 3 gün aralıklarla art arda T75 kültür şişelerinde hasat edilebilir . Izole makrofajların saflık ED-1 (Şekil 4), ED-3, OX-41 (Şekil 4) ve Iba1 9 gibi sıçan makrofaj-spesifik antikorların flowcytometry veya immünsitokimya tarafından değerlendirilir 95 ila% 99, izole hücreler polystylene boncuk aktif fagositoz (Şekil 5), rekombinant GM-CSF, LPS ile stimülasyon, ve çok çekirdekli dev hücreler 9 oluşumu üzerine inflamatuar / anti-inflamatuar sitokinlerin salgılanması proliferatif yanıt olarak makrofaj tipik özelliklerini gösterir. <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="Şekil 1" /> Şekil 1 sıçan karaciğer hücrelerinin karışık primer kültür makrofaj benzeri hücreler seçici izolasyonu ve saflaştırılması için bir şema. <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="Şekil 2" /> Şekil 2 sıçan karaciğer hücreleri ve makrofaj gibi hücrelerin çoğalması Primer kültür. Ok multinükleer dev hücreli gösterir. Ölçeği bar = 100 mikron. Elsevier izin İmmünolojik Yöntemleri Dergisi, Vol.360, 47-55 (2010) 9 yayımlanmaktadır. <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="Şekil 3" /> Şekil 3 sallayarak ve ek yöntemi makrofaj benzeri hücreler seçici izolasyon. Hücreler, matara sallayarak kültür ortamı içine askıya Daha sonra doku kültürü olmayan dereceli plastik tabaklar aktarılır ve 37 ° C&#39;de inkübe Kaplama gibi erken 10 dakika sonra, diğer bulaşıcı fibroblastik hücreler asılı kalırken, çanak yüzeye bağlı makrofaj benzeri hücreler. PBS ile iyice durulandıktan sonra, son derece saf bir makrofaj nüfus elde edildi. Bu hücreler kültür 40 dakika sonra tipik makrofaj morfolojisi kazanmıştır ve mitotik hücrelerin sık sık gözlenmiştir (oklar). Farklı kültür dönemlerde şişeler ele hücre sayıları daha sonra yapılan değişiklikler gösterilmiştir. Değerler üç beş şişeler ortalama ± SD. Deneyler, üç kez tekrarlandı ve veri farklı sembollerle gösterilir. Ölçeği bar = 100 mikron. Elsevier izin İmmünolojik Yöntemleri Dergisi, Vol.360, 47-55 (2010) 9 yayımlanmaktadır. <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="Şekil 4" /> Şekil 4 sıçan makrofajlar karşı monoklonal antikorlar ile makrofaj benzeri hücreler İmmünositokimyasal boyama. <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="Şekil 5" /> Şekil 5 makrofaj benzeri hücreler tarafından FITC etiketli mikro Fagositoz.

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

Tüm hayvanlar, hayvan deneyleri için Kurum Komitesi tarafından belirlenen kurallara ve düzenlemelere göre, Hayvan Sağlığı Ulusal Enstitüsü hayvan tesis tutulur ve ameliyat edildi.

Burada, yetişkin sıçan karaciğer hücrelerinin karışık primer kültürlerden gelen makrofajlar elde etmek için basit ve verimli bir yöntem raporu. Bizim sıçan karaciğer hücrelerinin karışık kültür yöntemi makrofajların roman proliferatif aktivite ilk güvenir ve daha sonra makrofajlar 9 olarak biyolojik özellikleri temelinde, bu hücrelerin sonraki izolasyonu ve saflaştırılması. Yetişkin karaciğer hücrelerinin karışık birincil kültür çoğaldı makrofajlar Kupffer veya parankimal hepatositler fraksiyonu kontamine ilişkili hücreler kaynaklanmış olabilir mümkün olabilir. Makrofajlar karışık birincil kültürlerde parankimal hepatosit 10 ve diğer fibroblastik hücreler dönüşüm neden olduğu çevresel değişikliklere yanıt olabilir . Sonuç olarak, yöntemi, gelişmiş donanım ve teknik becerileri kullanarak, sıçan karaciğer cellswithout birincil kültürünün yeterli sayıda ve saflık makrofaj gibi hücreleri izolasyonu sağlar. Buna ek olarak, yalıtım işlemi en fazla iki hafta boyunca aynı kültür şişesi ile tekrar edilebilir. Bu yöntem, diğer memeli türleri için geçerli olabilir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Sodyum pentobarbital çözüm </td><td> KYORITSU Seiyaku </td><td> Somnopentyl Enjeksiyon </td><td></td></tr><tr><td> Hank dengeli tuz solüsyonu (HBSS) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> Etilen glikol tetraasetik asit (EGTA) </td><td> Sigma-Aldrich </td><td> E-4378 </td><td></td></tr><tr><td> Kollajenaz </td><td> Wako Saf Chemical Industries, Ltd. </td><td> 032-10534 </td><td> Hücre disosiasyon derece (Lot kontrol gerekli) </td></tr><tr><td> Tripsin inhibitörü </td><td> Sigma-Aldrich </td><td> T-9128 </td><td></td></tr><tr><td> Eagle minimum esansiyel orta (MEM) </td><td> Sigma-Aldrich </td><td> M-4655 </td><td></td></tr><tr><td> Dulbecco&#39;nun değiştirilmiş bulunuyor Eagle orta (DMEM) </td><td> Sigma-Aldrich </td><td> D-6459 </td><td> Yüksek glukoz-tipi </td></tr><tr><td> Fetal buzağı serumu (FCS) </td><td> HyClone </td><td> SH30070.03 </td><td> Isı 56 ° C de 30 dakika süreyle inaktive. (Lot kontrol gerekli) </td></tr><tr><td> TrypLE Express </td><td> Invitrogen </td><td> 12605 </td><td></td></tr><tr><td> Dulbecco&#39;nun fosfat tamponlu salin (PBS) </td><td> Invitrogen </td><td> 14190 </td><td> Ca 2 +-Mg 2 + ücretsiz </td></tr><tr><td> ß-mercaptoethanol </td><td> Sigma-Aldrich </td><td> M-7522 </td><td> Stok: distile su içinde 100 mM </td></tr><tr><td> İnsülin </td><td> Sigma-Aldrich </td><td> I-5500 </td><td> Stok: 0,1 N HCl 10 mg / ml </td></tr><tr><td> Penisilin / streptomisin </td><td> Invitrogen </td><td> 15070 </td><td></td></tr><tr><td> Hücre süzgeç </td><td> BD Biosciences </td><td> 352360 </td><td> Mesh boyutu: 100 mikron </td></tr><tr><td> Doku kültürü şişesi </td><td> Sumitomo Bakalit Co, Ltd </td><td> MS-21250 </td><td> Yüzey alanı: 75 cm 2 </td></tr><tr><td> Sigara-doku kültürü plastik tabaklar </td><td> BD Biosciences </td><td> 351005 </td><td></td></tr><tr><td> Hücre kazıyıcı </td><td> Corning Inc. </td><td> 3010 </td><td></td></tr><tr><td> Karşılıklı çalkalayıcı </td><td> TAITEC </td><td> NR-1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

Kupffer hücreleri karaciğer özel ikamet eden makrofaj ve karaciğer 1-3 fizyolojik ve patolojik fonksiyonları önemli bir rol oynamaktadır. Karaciğer makrofajların izolasyon yöntemleri 4-6 iyi tanımlanmış olmasına rağmen, bu yöntemlerin çoğu böyle bir santrifüj elutriator ve teknik beceri olarak gelişmiş donanım gerektirir. Burada, karaciğer makrofajlar şematik olarak Şekil 1&#39;de gösterildiği gibi yetişkin sıçan karaciğer hücrelerinin karışık birincil kültürleri, yeterli sayıda ve saflık elde etmek için yeni bir yöntem sağlar. Iki adım perfüzyon yöntemi 7,8 karaciğer hücrelerinin disosiasyon sonra, çoğunlukla parankimal hepatositlerde oluşan bir kısmını hazırlanan ve T75 DMEM ve% 10 FCS.Parenchymal hepatositlerde oluşan kültür ortamı ile doku kültürü şişeleri içine numaralı seribaşı epitel hücre morfolojisi kaybedersiniz birkaç gün içinde bir kültür, yozlaşmış ya da fibroblast benzeri hücreler (Şekil 2) haline dönüştürmek. Kültür, yaklaşık 6 gün ilerledikçe, faz kontrast parlak, yuvarlak makrofaj benzeri hücreler (Şekil 2) fibroblastik hücre kağıda prolifere başlar. Gibi Makrofaj gibi hücrelerin büyümesini devam şişeyi yüzeyinde hücre sayfasını kapsayan ve yaklaşık 12 gün maksimum seviyelere ulaşmak. Kültür şişe çalkalanarak, makrofajlar kolaylıkla kültür ortamı içine askıya alınır. Gibi diğer kirlenmesine neden olan hücreleri askıda kalır makrofajlar seçici yapışma plastik tabaklar sonucu sonraki transfer ve kısa inkübasyon (Şekil 3). PBS ile birkaç durular sonra, ekli makrofajlar hasat edilir. 10&#39;dan fazla 6 hücre, iki haftadan daha uzun (Şekil 3), 2-3 gün aralıklarla aynı T75 doku kültürü şişesi sürekli hasat veya flow sitometri tarafından değerlendirilir olarak izole makrofajların saflık, 95 % 99 sıçan makrofaj-spesifik antikorların (Şekil 4) ile immünsitokimya izole hücrelerinin LPS ile uyarılması ve oluşumu üzerine polystylene boncuk aktif fagositoz (Şekil 5), GM-CSF rekombinant proliferatif yanıt, inflamatuar / anti-inflamatuar sitokinlerin salgılanması multinükleer dev hücreler 9. Sonuç olarak, bu yöntemi diğer memeli türleri için geçerli olabilir, karmaşık ekipman ve skills.This olmadan, yeterli sayıda ve saflık karaciğer makrofajlar elde etmek için basit ve etkili bir yöntem sağlar.

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Makrofajlar sıçan karaciğer hücrelerinin primer kültür elde etmek için yeni bir yöntem açıklanmıştır. Bu yöntem, plastik tabaklar, seçici eklenti kültür matara ve arıtma sallayarak kültürün yayılması, makrofajlar kullanır. Bu teknik, karmaşık ekipman ve becerileri verimli olmadan karaciğer makrofajlar sağlar.

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

a-simple-efficient-method-to-isolate-macrophages-from-mixed-primary

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

38.1K Görüntüleme.  National Institute of Agrobiological Sciences, Tsukuba, Japan. Makrofajlar sıçan karaciğer hücrelerinin primer kültür elde etmek için yeni bir yöntem açıklanmıştır. Bu yöntem, plastik tabaklar, seçici eklenti kültür matara ve arıtma sallayarak kültürün yayılması, makrofajlar kullanır. Bu teknik, karmaşık ekipman ve becerileri verimli olmadan karaciğer makrofajlar sağlar.

Video: A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

İlköğretim İnsan Keratinositler gelen Organotipik Radye Kültürlerin Üretimi

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

İmmünoloji ve Enfeksiyon

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Yenidoğan Rat Beyin Dokusu Karışık Glial Hücre Kültürleri Primer mikroglia İzolasyonu

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Akım Sitometri tarafından İnsan Nötrofiller Nükleer Faktör Aktivasyon Algılama Basit ve Verimli Yöntemi

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Kayma Stres İnsan Karaciğer Sinüzoidal Endoteline altında Koşullarına Lökositer İşe Alım Eğitim için Bir Akış Adezyon Deneyi

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Nucleofection İnsan THP-1 Makrofagların Yüksek Verimli Transfeksiyonu

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Fare Dendritik Hücre Alt Grupları İzolasyonunda bir Verimli ve Yüksek Verim Yöntem

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Basit ve Etkin Yaklaşım Mutant Vaccinia Virüs Vektörler Construct

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Fare Kemik İliği itibaren Üretimi ve GM-CSF tanımlanması Türetilmiş Alveoler gibi makrofajlar ve dendritik hücrelerin

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Fare Primer Hepatosit Kültürler bir inflamatuar yanıtın indüksiyon

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Conjunktiva&#39;dan Hücreleri İzole Etmek ve Fenotip Hedeflemek İçin İnvaziv Olmayan Bir Yöntem

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...