Yazarlar ifşa hiçbir şey yok.

PAAS Cam lameller 1. Hazırlık <ol><li> Düşük tüy bırakmayan mendil ile temizleyin 12 mm lamelleri. </li><li> Alev 12 mm lamelleri ve cımbız kullanarak Bunsen beki alevi geçirerek her 35 mm cam alt çanak kuyucuklarda 14 mm lamel. </li><li> Oda sıcaklığında 5 dakika boyunca 0.1 N NaOH, 200 ul mikro tedavi edin. </li><li> Aspire ve hava 30 dakika mikro kurulayın. </li><li> Çeker ocak içinde, oda sıcaklığında 10 dakika boyunca 3-aminopropiltrimetoksilan 50-100 ul mikro tedavi edin. Bu kimyasal plastik tepki gösterir; Bu nedenle, cam pipet kullanın ve çanak plastik ile temasını engellemek için tamamen mikro doldurmak değil. </li><li> 3-aminopropiltrimetoksisilan berrak hale gelene kadar yaklaşık olarak 10 dakika için ultra-saf su ile mikro-yıkayın. </li><li> Saf su iki kez sıkmak şişe kullanarak mikro durulayın. </li><li> Saf su, a 2 ml mikro yıkayın10 dakika boyunca orta hızda (yaklaşık 1 Hz) ayarlanmış bir rocker t oda sıcaklığı. </li><li> Aspire ve hava 30 dakika mikro kurulayın. </li><li> 30 dakika için orta hızda (yaklaşık 1 Hz) ayarlanmış bir rocker oda sıcaklığında 0.5% glutaraldehit solüsyonu 2 ml mikro tedavi edin. </li><li> 10 dakika boyunca orta hızda (yaklaşık 1 Hz) ayarlanmış bir rocker oda sıcaklığında, aşırı saf su, 2 ml ile mikro yıkayın. 30 dakikalık bir toplam iki kez daha tekrarlayın. </li><li> 30 dakika boyunca bir dik açıda (60 ° ya da daha yüksek) bir kuru mikro-. Not: Tabaklar bir desikatör içinde 2 ay boyunca saklanabilir. </li></ol> Invadopodia Tahliller için PAAS 2. hazırlanması <ol><li> Yumuşak PAA (% 8 akrilamid ve% 0.05 BIS) bir 1 ml çözelti için,% 40 akrilamid, 200 ul,% 2 BIS 25 ul ve ultra saf su 574 ul (Tablo 1) bir araya getirir. </li><li> Sabit PAA (% 8 akrilamid ve% 0.35 BIS) bir 1 ml çözelti için,% 40 akrilamid, 175 ve # 200 ul birleştirme181 l% 2 BİS ve ultra saf su 409 ul (Tablo 1). </li><li> Katı PAA (% 12 akrilamid ve% 0.60 BIS) bir 1 ml çözelti için,% 40 akrilamid, 300 ul,% 2 BIS 300 ul ve ultra saf su 169 ul (Tablo 1) bir araya getirir. </li><li> 15 dakika için çözümler degas. Sırasıyla, sert, yumuşak ve sert PAA çözümleri için, aşırı saf su içinde 1 mg / ml insan plazma fibronektinin 200, 215, ve 230 ul ekle. Tüm solüsyonlar için, 10 mg / ml akrilik asit, N-hidroksisüksinimid (NHS) ester 1 ul, 100 mg / ml amonyum persülfat 5 ul ve N, N, N &#39;, N&#39;-tetrametiletilendiamin (TEMED 2 ul Tablo 1). Nazik pipetleme ile karıştırın ve çözümleri kabarcıkları oluşturarak kaçının. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> PAA (1 mi) </td><td> % 40 AA (Ul) </td><td> % 2 (Ul) </td><td> Ultrapure Su (Ul) </td><td> 1 mg / ml FN (Ul) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS ester (Ul) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / ml APS (Ul) </td><td> TEMED (Ul) </td><td> Elastik Modül (Pa) </td></tr><tr><td> Yumuşak </td><td> 200 </td><td> 25 </td><td> 574 </td><td> 200 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Sert </td><td> 200 </td><td> 175 </td><td> 409 </td><td> 215 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Sert </td><td> 300 </td><td> 300 </td><td> 169 </td><td> 230 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22.692 </td></tr></tbody></table> Tablo 1: Bileşen VOLUmes ve invadopodia deneylerinde kullanılan, sert, yumuşak ve sert PAAS için elastik modüle sonuçlanır. AA = akrilamid, FN fibronektin, APS = amonyum persülfat =. <ol start="5"><li> Her kuyucuklarda merkezine üzerine PAA çözeltisi 8.48 ul pipetle. Not: Bu birim teorik kalınlığı 75 mikron bir PAA verir. </li><li> Yavaşça cımbız kullanarak kuyucuklarda olarak damlacık için 12 mm lamel Alevli tarafını düşük. </li><li> Sandviç PAA çözüm 15-30 dakika süreyle polimerize izin verin. Artık çözüm kontrol ederek polimerizasyonu doğrulayın. </li><li> Her yemeğin 1x PBS 2 ml ekleyin ve cımbız ile 12 mm lamelleri kaldırın. Not: 10x PBS laboratuarda yapılan ve 0.011 M KH 2 PO 4, 1.54 M NaCI, ve 0.056 M Na-2 HPO 4 oluşmaktadır. </li><li> 5 dakika boyunca oda sıcaklığında 1 x PBS, 2 ml ile mikro yıkayın. 15 dakikalık bir toplam iki kez daha tekrarlayın. </li></ol> 3. PÇekiş Gücü Tahliller için PaaS onarımı <ol><li> Yumuşak PAA (% 8 akrilamid ve% 0.05 BIS) bir 1 ml çözelti için,% 40 akrilamid, 200 ul,% 2 BIS 25 ul ve ultra saf su 566 ul (Tablo 2) bir araya getirir. </li><li> Sabit PAA (% 8 akrilamid ve% 0.35 BIS) bir 1 ml çözelti için,% 40 akrilamid, 200 ul,% 2 BIS 175 ul ve ultra saf su 401 ul (Tablo 2) bir araya getirir. </li><li> Katı PAA (% 12 akrilamid ve% 0.60 BIS) bir 1 ml çözelti için,% 40 akrilamid, 300 ul,% 2 BIS 300 ul ve ultra saf su 161 ul (Tablo 2) bir araya getirir. </li><li> 15 dakika için çözümler degas. Sırasıyla, sert, yumuşak ve sert PAA çözümlerine 1 mg / ml insan plazma fibronektinin 200, 215, ve 230 ul ekle. Sonikasyon 30 saniye için 200 nm kırmızı flüoresan mikroküreler (580/605 nm uyarım / emisyon) 8 ul. Her çözüm için, 200 nm kırmızı floresan mikroküreler 8 ul ekleyin10 mg / ml akrilik asit, NHS esteri 1 ul, 100 mg / ml amonyum persülfat 5 ul ve TEMED (Tablo 2) 2 ul. Nazik pipetleme ile karıştırın ve çözümleri kabarcıkları oluşturarak kaçının. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td style="width:40px"> PAA (1 mi) </td><td> % 40 AA (Ul) </td><td> % 2 BIS (Ul) </td><td style="padding-left: 4px; padding-right: 4px;"> Ultrapure Su (Ul) </td><td> 1 mg / ml FN (Ul) </td><td> Mikro-küreler (Ul) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS ester (Ul) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / ml APS (Ul) </td><td> TEMED (Ul) </td><td> Elastik Modül (Pa) </td></tr><tr><td> Yumuşak </td><td> 200 </td><td> 25 </td><td> 566 </td><td> 200 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Sert </td><td> 200 </td><td> 175 </td><td> 401 </td><td> 215 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Sert </td><td> 300 </td><td> 300 </td><td> 161 </td><td> 230 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22.692 </td></tr></tbody></table> Tablo 2:. Madde hacimleri ve çekiş kuvveti deneylerinde kullanılan, sert, yumuşak ve sert PAAS için elastik modüle ortaya çıkan AA = akrilamid, FN fibronektin, APS = amonyum persülfat =. <ol start="5"><li> Her kuyucuklarda merkezine üzerine PAA çözeltisi 8.48 ul pipetle. Not: Bu birim teorik kalınlığı 75 mikron bir PAA verir. </li><li> Yavaşça th Alevli tarafını düşürmekcımbız kullanarak kuyucuklarda olarak damlacık e 12 mm lamel. </li><li> Sandviç PAA çözüm 15-30 dakika süreyle polimerize izin verin. Artık çözüm kontrol ederek polimerizasyonu doğrulayın. </li><li> Her yemeğin 1x PBS 2 ml ekleyin ve cımbız ile 12 mm lamelleri kaldırın. </li><li> 5 dakika boyunca oda sıcaklığında 1 x PBS, 2 ml ile mikro yıkayın. 15 dakikalık bir toplam iki kez daha tekrarlayın. </li></ol> Invadopodia Tahliller için ECM 4. hazırlanması <ol><li> 37 ° C&#39;ye kadar jelatin solüsyonu (% 1 jelatin ve% 1 sukroz) ısıtın. </li><li> 45 ° açıyla cam alt yemekleri tutarken mikro altından dikkatlice aspire sonra 1 dakika boyunca jelatin çözeltisi 150 ul PAAS davranın. </li><li> 60 dakika boyunca kurutma optimize etmek için dik bir açı (60 ° veya daha yüksek), bir mikro-PAAS üzerindeki jelatin kalan ince bir tabaka kurutun. </li><li> I soğutulmuş bir% 0.5 glutaraldehid çözeltisi 2 ml mikro tedavi30 dakika boyunca oda sıcaklığında, ardından 15 dakika boyunca ce. </li><li> 5 dakika boyunca, bir 1x PBS 2 ml mikro yıkayın. 15 dakikalık bir toplam iki kez daha tekrarlayın. </li><li> 1 dakika süre ile, bir sodyum borohidrit solüsyonu 2 ml mikro tedavi edin. Yavaşça jelatin yüzeyinde oluşturan kabarcıklarını çıkarmak için bankta yemekleri dokunun. </li><li> Aspire 5 dakika için 1x PBS 2 ml mikro yıkayın. 15 dakikalık bir toplam iki kez daha tekrarlayın. </li><li> 15 dakika boyunca 4 ° C&#39;de 175,000 x g&#39;de 50 ug / 1x PBS ilave edildi ve santrifüj bir FITC-işaretlenmiş insan fibronektin çözeltisi ile seyreltilir. Ya piyasada mevcut etiketli fibronektin satın almak veya aşağıdaki gibi etiket: <ol><li> Diyaliz tüpünden borat tampon maddesi (170 mM sodyum metaborat tetrahidrat ve 40 mM NaCI) ve yerine 10 ml fibronektin 5 mg eritin. </li><li> Bir manyetik karıştırıcı üzerinde, oda sıcaklığında çözülmüş FITC 6 mg borat tamponu, 200 ml borunun yerleştirilmesi ile fibronektin Etiketkaranlıkta 1.5 saat için en düşük ayarlanmış ve. </li><li> İki hacim ile karanlık bir soğuk odada (4 ° C) &#39;de 3 gün boyunca düşük ayarlanmış ve bir manyetik karıştırıcı üzerinde 1 x PBS, 500 ml diyaliz günde değiştirir. </li><li> (1: 200) ile seyreltildi alikotları optik yoğunluğuna bağlı olarak hesaplayın FITC etiketli fibronektin konsantrasyonu 280 nm ve 493 nm olarak [OD 280 - (0.36 x OD 493)] / 1.4 kat 200 kat 2 seyreltme faktörü (hesaplarını mg / ml birimleri olarak elde edilen bir sonraki aşamada fibronektin konsantre gliserol) dir. </li><li> Gece boyunca 4 ° C de karanlık bir soğuk odada düşük ayarlanmış ve bir manyetik karıştırıcı üzerinde,% 50 gliserol çözeltisi, bir gece boyunca diyaliz). </li></ol></li><li> Karanlıkta 60 dakika boyunca oda sıcaklığında ve FITC ile işaretlenmiş fibronektin solüsyonundan 150 ul mikro tedavi edin. </li><li> Cam alt yemekleri tutarken dikkatli mikro altından FITC etiketli fibronektin çözüm aspire45 ° &#39;lik açı ve daha sonra sterilizasyon için karanlık bir hücre kültürü kaputu 10 dakika boyunca oda sıcaklığında% 70 etanol çözeltisi ile cam alt yemekler doldurun. Ayrıca cam alt çanak kapaklarını doldurmak veya% 70 etanol batırılmış düşük tüy bırakmayan mendil ile silin. </li><li> 5 dakika boyunca 1 x PBS ile doldurularak cam alt yemekler yıkayın. 15 dakikalık bir toplam 1x PBS ve 2 ml iki kez daha tekrarlayın. </li></ol> Invadopodia Tahliller 5. hazırlanması ve Görüntüleme <ol><li> (1: DMEM ve% 5 düşük-protein serumu,% 10 fetal sığır serumu ve taze eklenen epidermal büyüme faktörü 20 ng / ml ile aynı miktarda RPMI 1640 1) cam alt yemek ve invadopodia ortamının 2 ml 25,000 kanser hücrelerini ekleme gece inkübe. </li><li> Aspire hücreleri düzeltmek için 20 dakika süre ile karanlıkta, oda sıcaklığında% 3.7 paraformaldehid, 2 ml ile mikro tedavi etmek ve. </li><li> 1x PBS ve 2 ml iki pratik yıkamadan sonra, oda Tempe bir% 0.1 Triton X-100 çözeltisi, 2 ml ile mikro tedavi5 dakika süreyle karanlıkta rature hücreleri geçirgenliği. </li><li> 1x PBS içinde 2 ml bir hızlı yıkamadan sonra,% 3 sığır serum albümini 60 dakika hücreleri engellemek için karanlıkta, oda sıcaklığında bloke etme çözeltisi ile mikro-muamele. </li><li> 60 dakika boyunca oda sıcaklığında karanlıkta bir çözüm engelleme: fare anti-cortactin antikoru (750 1), 150 ul mikro inkübe edin. </li><li> 5 dakika boyunca 1x PBS 2 ml mikro yıkayın. 15 dakikalık bir toplam iki kez daha tekrarlayın. </li><li> (: 1: 500) ve 546 falloidinle (1: 750) 60 dakika süre ile karanlıkta, oda sıcaklığında çözelti bloke keçi 150 ul anti-fare antikoru 633 mikro inkübe edin. </li><li> 5 dakika boyunca 1x PBS 2 ml mikro yıkayın. 15 dakikalık bir toplam iki kez daha tekrarlayın. </li><li> Her kuyu doldurun ve 22 x 22 lamelleri ile karşılamak için montaj orta altı damla ekleyin. </li><li> Resim cortactin aktin 632/647 nm&#39;lik eksitasyon ve emisyon filtreleri kullanılarak invadopodia tespit etmek ve556/570 nm, sırasıyla, bir geniş alan ters mikroskop ile, yüksek NA 40X yağa batırılmış objektif kullanarak. Benzer şekilde, görüntü fibronektin 492/518 nm&#39;lik bir eksitasyon ve emisyon filtresi kullanılarak ECM bozulma tespit etmek. </li></ol> 6. Hazırlık ve Çekiş Gücü Tahliller Görüntüleme <ol><li> Cam alt yemekleri invadopodia ortamının 2 ml 15.000 kanser hücreleri ekleyin ve gece boyunca inkübe edilir. </li><li> Cam alt tabaklarında Kültür ortamı basınçla aynı takviyeleri (% 5 düşük-protein serumu,% 10 fetal sığır serumu ve taze eklenen epidermal büyüme faktörü 20 ng / ml), Önceden L-15 ortamına 2 ml ile değiştirin 37 ° C -warmed. </li><li> Bir ters mikroskop 1 saat boyunca yüksek nem ile 37 ° C&#39;de önceden dengelendi ile bir çevre odasında cam alt yemekler inkübe edin. </li><li> Hücresel pozisyonlarını işaretlemek için yüksek NA, otomatik evre bir geniş alan ters mikroskop 40X lens kullanarak görüntülerin dört setleri atın: <ol><li> Üzerine resim hücreleriFaz kontrast (&quot;faz&quot; resim) kullanarak PAAS p. </li><li> PAAS üstündeki mikro kürelerinin birinci tabaka kadar hücreler biraz altında odaklanarak 560/645 nm&#39;lik eksitasyon ve emisyon filtreleri kullanılarak görüntü floresan mikrosferleri odağa geldiği (görüntü &quot;stresli&quot;). </li><li> Buna ek olarak, (belirteç olarak küreleri kullanılarak) PaaS altına odaklanmak ve (&quot;alt&quot; görüntü) bir görüntü almak. Not: Bu resim amacı Örnek Sonuçlar tarif edildiği gibi çekme gücü hesaplamaları için her pozisyondaki gerçek PAA kalınlığı elde etmek için z-pozisyonunu tespit etmektir. </li><li> Bu görüntüler çoklu pozisyonlarda elde edilmiş sonra, yavaşça hücrelerin çıkarılması için% 10 Triton-X-çözeltisi 220 ul karıştırın. Daha önce tarif edildiği gibi (&quot;sıfır&quot; görüntü) her pozisyonda PAAS üstünde görüntü mikro-küreler. </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+density-induced+mechanoregulation+of+breast+cell+phenotype,+signaling+and+gene+expression+through+a+FAK-ERK+linkage.">Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.</a> Oncogene. 28, 4326-4343 (2009).</li><li>Munevar, S., Wang, Y., Dembo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+of+migrating+normal+and+H-ras+transformed+3T3+fibroblasts.">Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.</a> Biophysical journal. 80, 1744-1757 (2001).</li><li>Rosel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+Rho/ROCK+signaling+in+sarcoma+cells+drives+invasion+and+increased+generation+of+protrusive+forces.">Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.</a> Mol Cancer Res. 6, 1410-1420 (2008).</li><li>Indra, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+correlation+of+mechanical+forces+and+metastatic+capacity.">An in vitro correlation of mechanical forces and metastatic capacity.</a> Phys Biol. 8, 015015 (2011).</li><li>Parekh, A., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+invasiveness+by+the+physical+extracellular+matrix+environment.">Regulation of cancer invasiveness by the physical extracellular matrix environment.</a> Cell adhesion &amp; migration. 3, 288-292 (2009).</li><li>Weaver, A. M., Page, J. M., Guelcher, S. A., Parekh, A., Coutts, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology in Adhesion Protein Protocols. 1046, 171-189 (2013).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples).">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples).</a> Physics in medicine and biology. 52, 1565-1576 (2007).</li><li>Wang, J. H., Lin, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, 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=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, 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=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

Bu eser Ödülü Numarası K25CA143412 altında Ulusal Sağlık Enstitüleri Ulusal Kanser Enstitüsü (Parekh) tarafından desteklenmiştir. Biz ek destek Kulak Burun Boğaz Anabilim Dalı tarafından sağlanan olduğunu kabul etmek istiyorum. içerik sadece yazarların sorumluluğundadır ve mutlaka Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmemektedir.

Bu invaziv ve kontraktil hücresel davranış ilişkilendirmek için invadopodia ve çekiş kuvveti testleri için temel olarak kullanılabilir PAAS imal edilmesi için bir yöntem sunulmaktadır. PAAS, hücrelerin üzerindeki sertlik etkilerine bakmak ve çekme kuvvetlerinin 18,24,27 hesaplamak için kullanılmış olsa da, bu protokol matrise karşılık invazif ve kontraktil hücresel davranışları ilişkili aynı bükülmezliklerde PAAS göre paralel analiz geliştirme ilk mekanik özellikleri. Düzgün...

tümör ilişkili ECM sertliği aktomizin kasılma 1-3 artırarak malign davranış sürüş önemli bir faktör olarak tespit edilmiştir. Bu etki ilk olarak göğüs kanseri hücreleri ile gösterilmiştir, matris sertliği diğer kanser türü bir rol oynayabileceğini tümör sertliğini gösteren kanser 4-8 çeşitli türetilen hücre invaziv özelliklerini değiştirmek için bulunmuştur. Invaziv göç sırasında çapraz bağlı dokulara nüfuz etmek, kanser hücrelerinin fokal ECM 9 aşağılamak için proteinazı lokalize invadopodia olarak bilinen aktin-zengin yapışkan uzantıların kullanılması. Invadopodia invaziv hücrelerinin bir işareti olarak düşünülen ve tümör hücre istilası ve metastaz 10,11 implike edilmiştir. Önceki çalışma matris sertlik invadopodia numaralarını düzenleyen göstermiştir ve miyozin II faaliyet ve mechanosensitive proteinler 12 ile ECM bozulmasını 4,12 ilişkili olmuştur. VerilmişTümör yoğunluğu ve kanser saldırganlık 13,14 arasındaki korelasyon, bu sonuçlar kanser hücrelerinin aktomizin kontraktilitesinde yoluyla işgali ve metastaz sürücü sert tümör dokularına yanıt verebilir bir mekanizma öneririz. Nitro ECM sertlik ve in vivo doku yoğunluğunun kanser hücrelerinin 1,15-17 invaziv davranışını düzenleyen gösterilmiştir. Aktomizin kontraktilite, bu süreçte önemli görünmektedir iken, metastatik kapasite artışı ile ilişkilendirilmiştir ya da kasılma kuvvetleri 6,18-20 azalma olup olmadığı gibi mevcut çalışmalar çatışma. Ayrıca, bu güçlerin doğrudan invadopodia aktivitesini 21 arabuluculuk olmadığını bilinmemektedir. Biz son zamanlarda kanser hücresi kasılma kuvvetleri matris sertlik bağımlı ve invadopodia 5 ile ECM bozulma belirleyici olduğunu buldu. Bu sonuçlar hücresel kuvvetler invadopodia ac aracılık kanser ilerlemesinde önemli bir rol oynadığını düşündürmektedirtümör mikro-mekanik özelliklerine yanıt olarak bilirlik. Kanser hücrelerinin 5 invaziv ve kasılma özelliklerini ilişkilendirilmesi için, daha önceden sertliği bağımlı invadopodia aktivitesi 4,12,22 araştırmak için kullanılmıştır, farklı sertliklere sahip PAAS oluşturmak için bir protokol yapılması. Kimyasal PAAS boyunca insan plazma fibronektinin çapraz olarak, bu modifiye edilmiş hidrojeller hücreler iki deneyde 5 aynı katılıkları deneyimli sağlamak için invadopodia ve çekiş kuvveti deneyleri hem de baz olarak kullanılabilir. Invadopodia deneylerinde, fibronektin matris bozulmasını tespit PAAS için Kaplanmış ECM bağlantı jelatin bir doğal bağlanma alanı içerir. Çekiş kuvveti deneylerinde, fibronektin hücresel çekiş kuvvetleri hesaplamak için kullanılan mikroküreli değiştirmeleri tespit doğrudan hücresel yapışma için bir ligand sağlar. Biz sert, yumuşak adlandırdıkları Bu yöntem sonuçları,normal ve kanserli dokuların 23 rapor mekanik özelliklerinin aralığını kapsayan hangi Pa 5 1023, 7307, ve 22.692 arasında, cam alt yemekleri bağlı ve elastik modüle, E sahiptir sert PAAS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-Aminopropyltrimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic acid NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>A8060</td>
			<td>prepare fresh in fume hood 10 mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Life Technologies</td>
			<td>A22283</td>
			<td>can also use rhodamine</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>prepare fresh 10% solution in 1X PBS</td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>18606</td>
			<td>use six drops to fill microwells</td>
		</tr>
		<tr>
			<td>BIS (2%)</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td>make 3% for blocking solution in 1X PBS and store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9777</td>
			<td>pre-equilibrate in borate buffer for 15-30 min</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>use to make acrylic acid NHS ester solution</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PHARMCO-AAPER</td>
			<td>E200</td>
			<td>dilute with ultrapure water to 70%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30070.03</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Polysciences</td>
			<td>00639</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (35 mm coverslips)</td>
			<td>MatTek</td>
			<td>P35G-0-14-C</td>
			<td>coverslips are uncoated</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%)</td>
			<td>Polysciences</td>
			<td>01909</td>
			<td>dilute with 1X PBS to 0.5%</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa Fluor 633 antibody&#160;</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Life Technologies</td>
			<td>33016-015</td>
			<td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>PX-1565-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>mouse anti-cortactin 4F11 antibody&#160;</td>
			<td>EMD Millipore</td>
			<td>05-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>SX-0720-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>use to make 10X PBS stock and borate buffer</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>dilute with ultrapure water to 0.1 N</td>
		</tr>
		<tr>
			<td>Nu-Serum (low-protein serum)</td>
			<td>BD Biosciences</td>
			<td>355500</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros</td>
			<td>416785000</td>
			<td>typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td>
		</tr>
		<tr>
			<td>PBS (sterile)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>use for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Cellgro</td>
			<td>10-040-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>prepare fresh in fume hood 1 mg/ml in 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>sodium metaborate tetrahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0251</td>
			<td>use to make borate buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td>make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining</td>
		</tr>
	</tbody>
</table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

Katı tümör dokuları güçlü kanser hücresi göç ve istila düzenlenmesinde rol oynamaktadır. Çapraz-bağlanmış dokular içinden invaziv göç proteolitik olarak hücre-dışı matrisi (ECM) indirgeme invadopodia adı aktin zengin uzantılar ile kolaylaştırılır. Invadopodia aktivitesi sertliği sinyalleri aktomizin kontraktilite yoluyla bu hücre içi yapıları düzenleyebildiğini göstermektedir ECM sertliği ve kanser hücresi kasılma kuvvetleri bağlı olduğu gösterilmiştir. Kanser hücrelerinin invaziv ve kasılma özelliklerini farklı bükülmezliklerde poliakrilamid jeller (PAAS) göre invadopodia ve çekiş kuvveti testleri kullanılarak in vitro korele edilebilir. Kanser hücrelerinin invaziv ve kasılma özelliklerini farklı bükülmezliklerde poliakrilamid jeller (PAAS) göre invadopodia ve çekiş kuvveti testleri kullanılarak in vitro korele edilebilir. İki tahliller arasında bazı farklılıklar vardır, ancak burada sunulan protokol inci PAAS oluşturmak için bir yöntem temin etmektedirHer iki deneylerinde kullanılabilir ve kolayca kullanıcının belirli bir biyolojik ve teknik ihtiyaçlarına adapte edilebilir edilebilir.

Invadopodia tahlilinde, invadopodia tipik olarak hücre gövdesi içinde birkaç noktalı yapı ile aktin ve cortactin gibi belirteçler (Şekil 1) bir sınırlama ile tanımlanır. Hem aktif bozundurucu ve bozmaz invadopodia sayılabilir ve bu yapıların FITC etiketli fibronektin (Şekil 1) &#39;deki flüoresan sinyal eksik siyah alanlar ile lokalize edilip ayırt edilir. Invadopodia el sayılır ve hücre başına ECM bozulma el hücre hatları olan bu siyah alanlar eşikleme belirle...

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Tümörün mikro mekanik sertliği invadopodia aktivitesini artırarak ve kasılma actomyosin ile malign davranış sürüş önemli bir rol oynar. Kullanma poliakrilamid jelleri (PAAS) invadopodia ve çekiş kuvveti deneyleri bükülmezlik matris yanıt olarak kanser hücrelerinin invaziv ve kasılma özelliklerini incelemek için kullanılabilir.

Kanser Hücreleri üzerindeki Invadopodia ve Çekiş Gücü Tahliller için poliakrilamid jeller

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

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Medicine

12.0K Görüntüleme.  Vanderbilt University Medical Center.  Tümörün mikro mekanik sertliği invadopodia aktivitesini artırarak ve kasılma actomyosin ile malign davranış sürüş önemli bir rol oynar. Kullanma poliakrilamid jelleri (PAAS) invadopodia ve çekiş kuvveti deneyleri bükülmezlik matris yanıt olarak kanser hücrelerinin invaziv ve kasılma özelliklerini incelemek için kullanılabilir. 

Video: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Sıralama Kök Hücre Tahliller ve Akış Primer Beyin Tümörü Doku İşleme

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Kemirgen Ürogenital Sinüs mezenşimin ve İnsan Kök Hücre Doku Rekombinasyon kullanarak İnsan Prostat epiteli oluşumu

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Klinik ve Klinik Öncesi Araştırma Uygulamaları Tümör Hücre (CTC) Tahliller Sirkülasyon semiautomated adaptasyon

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Meme Kanseri Hücrelerinde östrojen düzenlenmiş microRNA profil

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Katı Örneklerinin From İlköğretim Yumurtalık Kanseri Hücreleri alma için yöntem

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

İnsan Hücre Lines Kemorezistans Yumurtalık Kanser Kök Hücreleri Zenginleştirme

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Primer Multipl Miyelom Hücrelerinin İlaç Duyarlılık Karakterizasyonu için bir Organotipik Yüksek Verimli Sistemi

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...