Name: polyacrylamide gels for invadopodia and traction force assays on cancer cells
Uploaded: 2015-01-04T15:00:00.000+00:00
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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><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 6 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 &amp;deg;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 &amp;deg;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</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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</td><td>EMD Millipore</td><td>05-180</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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</td></tr><tr><td>Sodium metaborate tetrahydrate</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 &amp;deg;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

Research

JoVE Journal

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

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Hücresel Daralma miktarının Şikayet Matrisler hazırlanması

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Biyomühendislik

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Çekme Kuvveti Mikroskopi Hücre Kültürü Yüzey civarında yerelleştirme raportör flöresan Beads için Yeni Bir Yöntem

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cerrahi Dokular İlköğretim İnsan Kolon Tümör Hücreleri İzolasyon ve Çekiş Sitometrinin Yumuşak Elastik Yüzeyler Doğrudan onları Kültürleme

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Basit Poliakrilamid-tabanlı Sertlik bağımlı Hücre Yanıtların Çalışmaları multiwell Sertlik Deneyi

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Doğrusal Invadozom Oluşumu ve Aktivitesi Çalışmak İçin 2D ve 3D Matrisler

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Hücre dışı matriks sertlik etkisi yapışık hücreleri bakteriyel enfeksiyon türleri çalışmak için çok iyi Format Polyacrylamide tabanlı tahlil

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

İmmünoloji ve Enfeksiyon

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Tek ve Çok hücreli Bağlamlarda Invadopodia aracılı Ekstrasellüler Matriks Proteoliz Kantitatif Ölçümü

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Biyoloji

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Çekiş Kuvveti Mikroskopisinde Uygulamalar için Hidrojel Desenleme Teknikleri Kullanılarak Hücre Yapışıklıklarının Kontrolü

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Mikrodesen Traksiyon Mikroskobu için Desen Oluşturma

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

Büyüme Konisi İlerlemesi için Actin Dynamics, Debriyaj Kavrama ve Çekiş Kuvveti Analizleri

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...