Bu çalışma, Smith Aile Biyomedikal Araştırma ve NIH RO1 AI083426-01 (FW) Mükemmellik Ödülü tarafından finanse edildi. Patrick Maschmeyer ve Melanie Flach Boehringer Ingelheim Vakfı doktora bursları tarafından desteklenmektedir.

C57BL / 6 fareler ~ 20 hafta veya daha büyük yaş kullanılmalıdır. 25-30g ağırlığındaki erkek farelerin kullanılması tavsiye edilir. Stellat hücrelerin hücre izolasyonu stellat önce 2 ay süreyle fareler A vitamini ile zenginleştirilmiş diyet beslenme verim artırılabilir. Balb / c farelerinde stellat hücreler verimi oldukça yüksek iken, yaklaşık 2x10 5 hepatik stellat hücreler, bir C57BL / 6 fare karaciğer arınmış olabilir. Aşağıdaki protokolü 5 farelere ayarlanır. Hayvan bakım ve deney onaylanan Kurumsal Hayvan Bakım ve Kullanım Kurulu protokolleri uyarınca yapıldı. 1. fare ciğeri in situ perfüzyon sindirim enzimleri <ol><li> 37 ° C su banyosunda SC1 tampon ve enzim perfüzyon çözümleri ısıtın. </li><li> Kanatlı bir infüzyon peristaltik bir pompa silikon tüp üzerine monte edin. </li><li> PBS ile pompa karaciğer in situ perfüzyon için kullanılacak olan akış hızı 6.5ml/min, laminer akış elde etmek için kalibre edin. SC1 çözümü ile silikon tüp dengelenmesi. </li><li> Ketamin (90 mg / kg) ve Xylazine çözümü ve derin bir uyuşturma sağlamak için reflekslerin kaybı için test (10 mg / kg) intraperitoneal enjeksiyonu ile fare anestezisi. </li><li> Uygun bir kaide üzerinde yatar pozisyonda fare sabitleyin. </li><li> Karın deride uzunlamasına bir kesi yapmak ve periton maruz makas ve forseps kullanın. </li><li> Periton dikkatlice açın ve portal ven göstermek için karın boşluğunun sol tarafında hayvan bağırsaklarında dışına taşımak. </li><li> Portal ven içine infüzyon kanülü takın. Bu adım için, bir stereomikroskopta kullanılması tavsiye edilir. </li><li> 30ml SC1 çözümü ile karaciğer perfüzyon başlayın. Vena kava inferiyor akışını başlatmak hemen sonra açın. Başarılı kızarma karaciğer, karaciğer doku renk kaybı ile gösterilir. </li><li> 30ml Pronase E çözümü ile karaciğer serpmek. Başarılı sindirim üzerine, karaciğer loblar şişmeye ve lobülleri kapsül ile farklı görünecektir. </li><li> 30ml kollajenaz P çözümü ile karaciğer serpmek. Bu noktada karaciğer şeklini kaybetmiş ve atonik ve şekilsiz görünüyor. </li><li> Diyafram ve çevre organların dikkatle karaciğer ayrı ve 70ml SC2 çözüm buz üzerinde saklayın. </li><li> Ek fareler için prosedürü tekrarlayın. </li></ol> 2. Fare ciğeri in vitro sindirim Ilave bütün adımlar bir laminer akış kaputu steril koşullar altında yapılmalıdır. <ol><li> Ciğeri keskin bir makas kullanarak yaklaşık 2x2x2mm 3 parçalar halinde kesin. </li><li> Pronase E-kollajenaz P çözüm 50ml ile karaciğer parçaları dahil olmak üzere 70ml SC2 askıya birleştirin ve DNaz I çözüm 1ml eklemek. </li><li> 37 20dk için ciğeri Digest ° C karıştırma. </li></ol> 3. Yoğunluk gradient santrifüj <ol><li> 6 50ml Falcon tüpler içine 70μm hücre süzgeçler ile hücre süspansiyonu Filtre ve SC2 tampon ile 50ml kadar ekleyin. 600g ve 4 ° C de 10 dakika süreyle Santrifüj </li><li> Süpernatantı dikkatlice 40ml aspirat, daha sonra her tüpe 150μl DNaz I çözümü eklemek ve hücreleri tekrar süspansiyon. </li><li> 4 50ml Falcon tüpler içine Havuzu hücre süspansiyonları ve GBSS-B, santrifüj, 600g ve 4 10 dakika süreyle yıkayın ° C </li><li> Pelet ve her tüp eklemek 150μl DNaz I çözüm rahatsız etmeden dikkatlice aspire kadar mümkün olduğunca süpernatant. 10ml tüp başına GBSS-B hücre pelet yeniden süspanse edin. </li><li> Havuz 2 50ml Falcon tüpler içine hücre ve tüp başına 36ml toplam hacmi GBSS-B. Her bir tüp 14ml Nycodenz çözümü ekleyin ve iyice karıştırın. </li><li> Toplam 10 tüp sonuçlanan hücre süspansiyonu, 10ml 12ml degrade santrifüj tüpü içine aktarın. 1.5ml GBSS-B için tüp yavaşça hücre süspansiyonu kaplaması. </li><li> 1500g ve 4 15dk için gradyanlar Santrifüj ° C frensiz. Daha sonra ise stellat hücreleri, beyaz bir halka olarak interfaz bulunan, hepatositler tüpün alt kısmında pelet olacaktır. </li><li> Stellat hücreler içeren interfaz dikkatlice hasat ve GBSS-B ile yıkayın; 600g ve 4 santrifüj 10dk ° C </li><li> Süpernatantı aspire ve% 10 ısı inaktive FBS,% 1 Penisilin / Streptomisin, 2mm L-Glutamine, 1mm sodyum piruvat ve 10mM Hepes ile desteklenmiş 20ml DMEM hücrelerin tekrar süspansiyon haline getirin. 2x10 4 hücre / cm 2 &#39;lik bir konsantrasyon hücrelerin doku kültürü şişeler içine aktarın. 37 ° C ve% 5 CO 2 hücreleri inkübe edin. </li><li> Medya hepatik stellat hücreleri kısa bir süre (yaklaşık 2 saatlik hazırlıktan sonra) yapışık ölü hücreleri ve hücre artıkları yıkayın olarak değiştirin. </li><li> Ertesi gün, stellat hücres perinükleer A vitamini depolama lipid veziküller ile karakteristik yıldız şeklinde morfoloji geliştirmelidir. </li><li> Sonraki deneyler için, hepatik stellat hücreler Accutase, örneğin hafif bir enzimatik çözümlerini kullanarak kaplı olmayan plastik yüzeylere kolayca ayrılabilir </li></ol> 4. Temsilcisi Sonuçlar: Sağlanan protokolü kullanılarak hepatik stellat hücreler hazırlanmasını takiben, izole nüfusun saflık glial fibriller asidik protein yıldıza benzer şekil, perinükleer lipid damlacıkları ve anlatım (GFAP gibi bu hücre tipi üç temel özellikleri göz önüne alınarak test edilebilir .) Hücre izolasyonunu takiben hepatik stellat hücreler 2s karakteristik bir görünüm yanı sıra, in vitro kültüründe 1 ve 3 gün Temsilcisi resimler Şekil 1&#39;de tasvir edilmektedir . Şekil 2, hepatik stellat hücreler, hücre izolasyonu takiben 3 gün süreyle kültüre GFAP için bir temsilci immünofloresan boyama gösterir. Hepatik stellat hücreler in vitro kültür sırasında miyofibroblastik hücrelerine ayırt . Bu aktive stellat hücrelerin karakteristik damgasını alfa düz kas aktin (αSMA) ifadesidir. Şekil 3 in vitro kültüründe gün 7 stellat hücreleri aktive ASMA ve miyozin IIA immünofloresan boyama gösterir . <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="Şekil 1" /> Şekil 1 hepatik stellat hücreler karakteristik morfolojisi. Stellat hücreleri kullanarak fare ciğeri sağlanan protokol izole edildi. Görüntüleri stellat hücreler hücre izolasyonu sonra 2s (a, b) yanı sıra, günde 1 (c) ve 3 (d) in vitro kültüründe gün betimliyor . Hepatik stellat hücreler yüksek miktarda lipid veziküllerin perinükleer sitelerinde sergilerler ve in vitro kültüründe ilk gün (Büyütme 200x) sırasında kendi ayırt edici astral gibi morfoloji kazanmak . <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="Şekil 2" /> Şekil 2 Karaciğer stellat hücreler, özellikle karaciğer GFAP ifade . Stellat hücreler, in vitro kültüründe gün 3 izole ve immunofluorescently GFAP (kırmızı) için boyandı. Hücre çekirdekleri mavi (leke Hoechst) tasvir edilmektedir. <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="Şekil 3" /> Şekil 3. Karaciğer stellat hücreler miyofibroblastlar farklılaşırlar. 7 gün boyunca C57BL / 6 farelerinde izole hepatik stellat hücreler kültüre edildi. Daha sonra, odasına slaytlara aktarılır ve ASMA (kırmızı ile gösterilen) ve miyozin IIA (yeşil tasvir) için boyandı. Hücre çekirdekleri Hoechst (mavi) ile zıt. <table border="1"><tbody><tr><td colspan="2"> SC1 Tampon </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td> Glikoz </td><td> 900mg </td></tr><tr><td> Hepes </td><td> 10 ml 1M stok solüsyonu </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Fenol Kırmızı </td><td> 6mg/dl </td></tr><tr><td> dH 2 O </td><td> 1l için doldurmak </td></tr><tr><td colspan="2"> SC2 Tampon </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 560mg </td></tr><tr><td> Hepes </td><td> 1M stok solüsyonu, 10ml </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Fenol Kırmızı </td><td> 6mg/dl </td></tr><tr><td> dH 2 O </td><td> 1l için doldurmak </td></tr><tr><td colspan="2"> GBSS-A Tampon </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glikoz </td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Fenol Kırmızı </td><td> 6mg/dl </td></tr><tr><td> dH 2 O </td><td> 1l için doldurmak </td></tr><tr><td colspan="2">GBSS-B Tampon </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glikoz </td><td> 991mg </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Fenol Kırmızı </td><td> 6mg/dl </td></tr><tr><td> dH 2 O </td><td> 1l için doldurmak </td></tr><tr><td colspan="2"> Pronase E Perfüzyon Çözüm </td></tr><tr><td> Pronase D </td><td> 100mg (4000 PU / mg dak.) </td></tr><tr><td> SC2 Tampon </td><td> 200ml </td></tr><tr><td colspan="2"> Kollajenaz P Perfüzyon Çözüm </td></tr><tr><td> Kollajenaz P </td><td> 85mg (1.78 U / mg LYO) </td></tr><tr><td> SC2 Tampon </td><td> 200ml </td></tr><tr><td colspan="2"> Pronase E-kollajenaz P Çözüm </td></tr><tr><td> Pronase D </td><td> 50mg (4000 PU / mg dak.) </td></tr><tr><td> Kollajenaz P </td><td> 85mg (1.78 U / mg LYO) </td></tr><tr><td> SC2 Tampon </td><td> 50ml </td></tr><tr><td colspan="2"> DNaz I Çözümü </td></tr><tr><td> DNaz I </td><td> 6 mg (yaklaşık 2000U/mg) </td></tr><tr><td> GBSS-B </td><td> 3ml </td></tr><tr><td colspan="2"> Nycodenz Çözüm </td></tr><tr><td> Nycodenz </td><td> 8g </td></tr><tr><td> GBSS-A </td><td> 28ml </td></tr></tbody></table> Tablo 1. Tamponlar ve hepatik stellat hücrelerin izolasyonu için gerekli enzim çözümleri. Tüm tamponları pH 7,3-7,4 ayarlanmış olmalıdır. Ayrıca, tüm tamponlar steril filtrasyon tavsiye edilir. Verilen enzimatik aktivite göre kullanılan enzim miktarı uyum önemlidir.

<ol>
	<li>Friedman, 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=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

Karaciğer stellat hücreler karaciğer temel fizyolojik ve patofizyolojik süreçleri düzenler. Ayrıca, stellat hücreler karaciğer bağışıklık yanıtının önemli bir bileşeni render, antijen sunan özelliklere sahip. Hepatik stellat hücreler karaciğer toplam hücre sayısı% 10-15 oluşturan rağmen, bu hücrelerin izolasyonu Disse perisinüzoidal uzayda lokalizasyonuna nedeniyle zordur. Burada yerinde sindirim ve sonraki gradient santrifüj fare ciğeri hepatik stella...

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog No. </th></tr><tr><td> 70μm Hücre Süzgeç </td><td> BD </td><td> 352350 </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> Sigma-Aldrich </td><td> C3306 </td></tr><tr><td> Kollajenaz P </td><td> Roche </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11960 </td></tr><tr><td> DNaz I </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21-031-cv </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> Fetal sığır serumu </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Glikoz </td><td> Sigma-Aldrich </td><td> G7528 </td></tr><tr><td> Gradient Santrifüj Tüpleri </td><td> Greiner Bio-One </td><td> 163160 </td></tr><tr><td> Hepes </td><td> Gibco </td><td> 15630 </td></tr><tr><td> KCL </td><td> Sigma-Aldrich </td><td> P9541 </td></tr><tr><td> KH 2 PO 4 </td><td> Sigma-Aldrich </td><td> P9791 </td></tr><tr><td> L-Glutamine </td><td> Gibco </td><td> 25030 </td></tr><tr><td> MgCl 2 x 6H 2 O </td><td> Fluka </td><td> 63068 </td></tr><tr><td> MgSO 4 x 2 O 7H </td><td> Sigma-Aldrich </td><td> M2773 </td></tr><tr><td> Na 2 HPO 4 x 2H 2 O </td><td> Fluka </td><td> 71643 </td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S3014 </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> Fluka </td><td> 71507 </td></tr><tr><td> NaHCO 3 </td><td> Fluka </td><td> 71628 </td></tr><tr><td> Nycodenz </td><td> Accudenz AG </td><td> AN7050/BLK </td></tr><tr><td> Penisilin / Streptomisin </td><td> Gibco </td><td> 15140 </td></tr><tr><td> Peristaltik Pompa </td><td> Cole Parmer </td><td> HV-7523-70 </td></tr><tr><td> Fenol Kırmızı </td><td> Sigma-Aldrich </td><td> P4633 </td></tr><tr><td> Pronase D </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> Silikon Tüp </td><td> Masterflex </td><td> HV-96.440-14 </td></tr><tr><td> Sodyum Piruvat </td><td> Gibco </td><td> 11360 </td></tr><tr><td> Doku kültürü şişesi (25cm 2) </td><td> BD </td><td> 353108 </td></tr><tr><td> Kanatlı İnfüzyon Seti </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

Karaciğer yıldız gibi morfoloji stellat hücreleri, karaciğer yerleşik hücreleri ve karaciğer sinüzoidal endotel hücreleri ve hepatositlerde 1,2 arasında Disse bir boşluk bulunmaktadır . Stellat hücreler, kemik iliği öncüleri türetilen ve toplam vücut A vitamini 1, 2% 80 kadar mağaza vardır . Stellat hücrelerin aktivasyonu üzerine, böylece karaciğer fibrozu 3 katkıda miyofibroblastlar içine ekstraselüler matriks üretmek için ayırt. Sözleşme yeteneklerini dayanarak, miyofibroblastik stellat hücreleri, portal hipertansiyon 4 ile ilişkili vasküler tonunu ayarlayabilir . Son zamanlarda, hepatik stellat hücreler güçlü antijen sunan hücreler ve NKT hücrelerinin yanı sıra, geleneksel T lenfositleri 5 etkinleştirebilirsiniz gösterdi. Burada, fare karaciğer hepatik stellat hücrelerinin etkin bir şekilde hazırlanması için bir yöntem mevcut. Perisinüzoidal yerelleştirme nedeniyle, hepatik stellat hücreler izolasyonu çok adımlı bir süreçtir. Stellat hücreler Disse uzay izolasyon erişilebilir kılmak için, fare ciğeri, sindirim enzimleri Pronase E ve kollajenaz P. ardından perfüzyon ile in situ perfüze olan, karaciğer doku Pronase E ve kollajenaz P ile ek enzimatik muameleye tabi tutulur in vitro. Daha sonra, bu yöntem büyük miktarda A vitamini depolama hepatik stellat hücreler lipid damlacıkları yararlanır. Bu özellik,% 8 Nycodenz degrade santrifüj diğer karaciğer hücre tipleri stellat hücrelerin ayrılmasını sağlar. Burada açıklanan protokol stellat hücreler son derece saf ve homojen bir nüfus verir. Hazırlıkları Saflık floresan mikroskobu veya flow sitometri analizi öncesinde marker molekül glial fibriller asidik protein (GFAP), boyama ile tespit edilebilir. Ayrıca, ışık mikroskobu, yüksek miktarda lipid damlacıklarının limanı yıldız şeklinde hepatik stellat hücreler benzersiz bir görünüm ortaya koymaktadır. Birlikte ele alındığında, biz temsilcisi morfolojik görünüm görüntüleri ve stellat hücre varlık tanımlamak için yardımcı GFAP ifade de dahil olmak üzere hepatik stellat hücreler, verimli izolasyonu için ayrıntılı bir protokol mevcut.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

Burada fare karaciğer hepatik stellat hücrelerin izolasyonu için bir yöntem açıklanmaktadır. Stellat hücre saflaştırma için, fare ciğeri sindirilir<em&gt; In situ</em&gt; Ve<em&gt; In vitro</em&gt; Yoğunluk gradiyenti santrifüj önce pronase kollajenaz tedavi. Bu teknik son derece saf hepatik stellat hücreler elde edilir.

Hücreler stellat Yedi Adımda

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

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Immunology and Infection

33.5K Görüntüleme.  Harvard Medical School. Burada fare karaciğer hepatik stellat hücrelerin izolasyonu için bir yöntem açıklanmaktadır. Stellat hücre saflaştırma için, fare ciğeri sindirilir In situ Ve In vitro Yoğunluk gradiyenti santrifüj önce pronase kollajenaz tedavi. Bu teknik son derece saf hepatik stellat hücreler elde edilir.

Video: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Tüm Yaprak Görüntüleme kullanarak Bakteriyel Hücreler Live Stoma Tepki Değerlendirilmesi

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

İmmünoloji ve Enfeksiyon

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Su bazlı Algılamak için Teğet Akış Hollow-fiber Ultrafiltrasyon ve Isı Disosiyasyon Adımlar kullanır A Modifiye EPA Method 1623<em&gt; Cryptosporidium</em&gt; Ve<em&gt; Giardia spp.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Naif CD4 adenoviral İletimi Treg Farklılaşma Eğitim Hücreler T

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Bir Antijen odaklı Kolit Modelinin Geliştirilmesi Antijen T hücreleri için Hücreler sunulması ile Antijen Sunumu Eğitim için

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Hassas kesimli Canlı Pulmoner Dendritik Hücreler Visualize&#39;a Fare Akciğer Dilimleri

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Hızla Nötrofil Kemotaksis Analizinde Doğrudan Bir Damla Kan Alınacak Bütünleşik Çip Yöntemi

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Geliştirme ve doğrulama vericiyi bir tek molekül dijital enzim bağlı Immunosorbent Assay insan Interferon-α için dizi

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...