Yazarlar DMF cihazları ve DMF merkezi tasarım ve geliştirme katkıları için Ronald F. Renzi ve Michael S. Bartsch teşekkür ederim. Bu araştırma tam Sandia Ulusal Laboratuarlarında Laboratuvarı Yönetmen Araştırma ve Geliştirme Programı tarafından desteklenmiştir. Sandia sözleşme DE-AC04-94AL85000 altında Enerji Ulusal Nükleer Güvenlik İdaresi ABD için Sandia A.Ş., Lockheed Martin Corporation&#39;ın iştiraki, tarafından yönetilen ve işletilen bir çok programı laboratuvardır.

Bu genel bir protokol çalışmaları geniş bir yelpazede için platformun uygulamayı desteklemek için tasarlanmıştır, bu raporda açıklanan temsilcisi çalışma özgü yönleri parantez içinde dışarı ayrılır Şekil 2 protokolü kullanılarak gerçekleştirilen temsili bir çalışma göstermektedir.. Protokolde, tüm &quot;talimat&quot; komutları önceden belirlenmiş komut kullanımı ile otomatik unutmayın. Adım 2 Adım 1 (Adım 1.7 ve 1.8 sırasında örneğin) paralel olarak yapılabilir bu da unutmayın ve bu adım 2.1 genellikle atlanır (desenli / kaplı DMF cihaz levhalar yıkanır ve tekrar kullanılabilir, çünkü; Adım 5.2) . 1. Hücre Perfüzyon Odalar montaj ve doldurmak <ol><li> Coat sterilize (otoklav) mikrokapilerler (erimiş-silis 15 cm uzunluğunda,, 679 mikron od, 534 mikron id) iç yüzeyleri steril fibronektin solüsyonu (50 mg / ml 30 ul) çizmek için şırınga pompası talimat ile fibronektin ile, her birinemikrokılcal, (2 saat boyunca 37 ° C) kuluçkaya çözeltiyi çekmek için bir şırınga pompası talimat ve mikrokapilerler kuru (6 saat boyunca oda sıcaklığında (RT)) izin vermek. </li><li> CapTite parçaları (6 perfüzyon odaları, 8-port şırınga pompası) kullanarak, polikarbonat tüp (500 mikron od, 100 mikron id) ve çok portlu şırınga pompası için boru her fibronektin kaplı microcapillary (perfüzyon odası) bağlayın. </li><li> Bant kullanarak, istenilen sıcaklığa ayarlanmış bir ısı bloğu (37 ° C) her perfüzyon odasının vücut sabitleyin. </li><li> Bir rezervuar içinde perfüzyon odaları arasında açık uçları (de microcentifuge tüp veya mikrotitre plaka) taze büyüme ortamı içinde süspansiyon haline içeren hücreler (P388D.1 sıçangil makrofaj) (RPMI-1640,% 10 FBS, 500 ünite / ml penisilin, 500 ug daldırın / istenilen konsantrasyonda (10 6 hücre / ml), streptomisin ml &#39;de). </li><li> Yeterli ai ardından her perfüzyon odasına hücreleri (10 ul, 10 4 hücre) çizmek için şırınga pompası talimatr ısı bloğuna güvenli bölümüne sıvı fişi (~ 4.5 cm) taşımak için. </li><li> Hücrelerin perfüzyon odaları arasında fibronektin kaplı iç yüzeyleri (-, 2 saat 37 ° C&#39;de 1) uyması için izin verir. Bu arada, hücreler rezervuar taze büyüme ortamı içeren yeni bir rezervuara perfüzyon odaları arasında açık uçları aktarın. </li><li> Uyum döneminden sonra, atık taze orta (10 ul) geri ve yapıştırılır üzerinde yeni sıvı fişleri (taze orta) konumlandırmak için yeterli hava ile takip sıvı fişleri (klimalı ortam ve serbest hücreler) göndermek için şırınga pompası talimat hücreler. </li><li> Hücre popülasyonlarının (- ~ 1.000 hücre / odasının mikro ölçekli kültür oluşturulan nüfus 24 saat 16) yeterince dengelenir ve genişletilmiş kadar düzenli aralıklarla (her 2 saat) orta değişim (yani tekrar Adım 1.7) yürütmek için devam edin. </li></ol> 2. DMF Cihaz imal ve Hub Mimarlık monte <ol><li&gt; olarak kırk altı indiyum kalay oksit (ITO) fotolitografi ve aşındırma ile elektrotlar (damlacık harekete sürücüleri), ve 5 Parylene-C mikron ve 50 ile kat ile DMF cihazın alt plaka, daha önce 14,29 açıklanan desen Teflon-AF nm. Yukarıda olduğu gibi, kaplama ile Teflon AF ile desensiz İTO cam alt tabakası DMF cihazın üst plakanın oluşturulması. </li><li> Bu aralık, çalıştırma elektrot boyutu 2,5 mm (2) ile bir araya damlacık hacmi (2.5 ul tanımlar, metal sıkıştırma kareler kullanılarak, plakalar arasındaki bir 400 mikron aralığı korumak girintiler sahip bir polimer döküm 14,30 içine DMF levhaları sabitlemek elektrot başına). DMF montaj basit yollarla 24 gerçekleştirilebilir unutmayın. </li><li> Ekle Teflon kaplı transferi mikrokapilerler (360 mikron od, 100 mikron id; 3.5 - 4.0 cm uzunluğunda) DMF plakalar arasındaki boşluğa, kendi soydaş harekete elektrot kenarına uzatmak için her konumlandırma. </li><li> Ters trher transfer microcapillary yapıştırmayın bir CapTite uygun d. </li><li> İTO elektrotlara voltaj sağlamak için elektrik bağlantıları meşgul. Elektrot aktivasyon dizileri önceden belirlenmiş komut çalıştıran bir bilgisayar kontrollü elektronik arayüzü kullanılarak otomatik vardır. </li><li> İsteğe bağlı: damlacıkların izleme kolaylaştırmak için bir şarj bağlı cihaz (CCD) kamera (DCR-HC96 (Sony, Japonya)) ile donatılmış bir mikroskop öteleme aşamasında (SZ-6145TR (Olympus, Japonya)) üzerine monte DMF merkezi Taşı . 31 </li></ol> 3. DMF Hub için Perfüzyon Odalar bağlayın ve Hücre Nüfus teşvik <ol><li> Orta hazneden perfüzyon odaları açık uçları (Adım 1.6 bakınız) çıkarın ve DMF merkezi devri mikrokapilerler (Adım 2.4) ucuna yapıştırılmış CapTite parçaları içine yerleştirin. </li><li> Atık perfüzyon odalarında sıvı fişleri (klimalı ortam) göndermek için şırınga pompası söyleyin. </li><li> Öğrbir on-board rezervuar (E. coli% 0.1 Pluronic F 127 a / h taze ortamda 100 mg / ml &#39;de BioParticles) uyaran çekmek ve her transfer microcapillary uygun boyutu (10 ul) bir damlacık sunmak için DMF merkezi ruct . On-board rezervuarları silindirik şekilli bölmeleri (1.5 x 1.5 cm her biri) oluşan ve boru ile DMF Hub bağlanır. </li><li> Uyaran transfer mikrokapilerler takılan çekmek ve perfüzyon odalarında hücre popülasyonlarının üzerinde fişler konumlandırmak için yeterli hava ile takip etmek şırınga pompası söyleyin. </li><li> Uyarıcı hücreler ile (-, 4 saat 37 ° C&#39;de 1 için) inkübe edin. İsteğe bağlı: düzenli aralıklarla taze uyaran (yani Adımları 3.2-3.4) için değişimi. </li></ol> 4. Uyarım sona ve Analiz için Hücre Lizatları Kurtar <ol><li> İsteğe bağlı: bir post-stimülasyon süresi gerekiyorsa, (yani Adımları 3.2 taze orta uyarıcı içeren sıvı fişler alışverişi - 3.4,) Teşvik için taze orta yerine ve inkübasyon ve gerektiğinde alışverişi devam ediyor. </li><li> Değişimi yıkama tamponu (Prelüd Direkt modülü Lizis Tampon B) (yani Adımları 3,2-3,4, uyarıcı için yıkama tamponu yerine) için perfüzyon odaları içindeki sıvı fişleri, ve benzeri gibi (5 dakika) tabi inkübe edin. </li><li> Döviz lizis çözüm için sıvı fişleri (tampon yıkama) (Prelude Doğrudan Lizis Modülü Tampon 0,1% Pluronik F 127 a / h ile) (yani tekrar Adımlar 3,2-3,4, teşvik için lizis çözüm yerine), ve inkübe gerektiği gibi (5 dakika) . </li><li> DMF merkezi için sıvı fişleri (hücre lizatları) göndermek için şırınga pompası söyleyin. </li><li> DMF merkezi sökün, DMF cihazı (bu damlacık karıştırma neden olabilir gibi, yan plaka yatırmak için dikkat kullanarak) üst plaka kaldırmak ve off-platformu analizi için bir Pipetman veya şırınga kullanarak lizatları (gen ekspresyon profili toplamak QPCR aracılığıyla). </li></ol> 5. Için temiz Platformu DonanımYeniden kullanmak <ol><li> Oda sıcaklığında 10 dakika boyunca% 10 çamaşır transferi mikrokapilerler ve CapTite parçaları (su, v / v) batırmak, azot gazı kullanılarak iyonu giderilmiş su ve kuru ile durulayın. </li><li> Oda sıcaklığında 10 dakika boyunca% 10 çamaşır DMF cihaz plakaları daldırın, izopropanol ile iyice yıkayın deiyonize su ile devam eder ve kuru bir nitrojen gazı kullanılarak 10 dakika boyunca 160 ° C&#39;de pişirme izledi. </li><li> Şırınga pompa polikarbonat tüp ve DMF cihazın polimer döküm ve sıkıştırma çerçeve, temizlik olmadan yeniden kullanılabilir. Perfüzyon odasının mikrokapilerler her bir deney sonra atılır. </li></ol>

<ol>
	<li>Young, E. W. K., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microfluidic+cell+culture+in+controlled+microenvironments.">Fundamentals of microfluidic cell culture in controlled microenvironments.</a> Chemical Society Reviews. 39, 1036-1048 (2010).</li><li>Yeo, L. Y., Chang, H. C., Chan, P. P. Y., Friend, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+bioapplications.">Microfluidic devices for bioapplications.</a> Small. 7, 12-48 (2011).</li><li>Zhang, B., Kim, M. C., Thorsen, T., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-contained+microfluidic+cell+culture+system.">A self-contained microfluidic cell culture system.</a> Biomedical Microdevices. 11, 1233-1237 (2009).</li><li>Skafte-Pedersen, P., 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=A+self-contained,+programmable+microfluidic+cell+culture+system+with+real-time+microscopy+access.">A self-contained, programmable microfluidic cell culture system with real-time microscopy access.</a> Biomedical Microdevices. 14, 385-399 (2012).</li><li>Barbulovic-Nad, I., Au, S. H., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+platform+for+complete+mammalian+cell+culture.">A microfluidic platform for complete mammalian cell culture.</a> Lab Chip. 10, 1536-1542 (2010).</li><li>Zhou, Y., Pang, Y., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Openly+Accessible+Microfluidic+Liquid+Handlers+for+Automated+High-Throughput+Nanoliter+Cell+Culture.">Openly Accessible Microfluidic Liquid Handlers for Automated High-Throughput Nanoliter Cell Culture.</a> Analytical Chemistry. 84, 2576-2584 (2012).</li><li>Wang, H. Y., Bao, N., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+cell+array+with+individually+addressable+culture+chambers.">A microfluidic cell array with individually addressable culture chambers.</a> Biosensors and Bioelectronics. 24, 613-617 (2008).</li><li>Cimetta, E., Cagnin, S., Volpatti, A., Lanfranchi, G., Elvassore, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+culture+of+droplet-confined+cell+arrays.">Dynamic culture of droplet-confined cell arrays.</a> Biotechnology Progress. 26, 220-231 (2010).</li><li>Hung, P. J., Lee, P. J., Sabounchi, P., Lin, R., Lee, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+perfusion+microfluidic+cell+culture+array+for+high-throughput+cell-based+assays.">Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.</a> Biotechnology and Bioengineering. 89, 1-8 (2005).</li><li>King, K. R., 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=A+high-throughput+microfluidic+real-time+gene+expression+living+cell+array.">A high-throughput microfluidic real-time gene expression living cell array.</a> Lab on a Chip. 7, 77-85 (2007).</li><li>G&#243;mez-Sj&#246;berg, R., Leyrat, A. A., Pirone, D. M., Chen, C. S., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile,+fully+automated,+microfluidic+cell+culture+system.">Versatile, fully automated, microfluidic cell culture system.</a> Analytical Chemistry. 79, 8557-8563 (2007).</li><li>Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+-+Putting+electrowetting+to+work.">Chemistry - Putting electrowetting to work.</a> Science. 322, 539-540 (2008).</li><li>Gong, J., Kim, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-electronic+droplet+generation+on-chip+with+real-time+feedback+control+for+EWOD+digital+microfluidics.">All-electronic droplet generation on-chip with real-time feedback control for EWOD digital microfluidics.</a> Lab Chip. 8, 898-906 (2008).</li><li>Choi, K., 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=Integration+of+field+effect+transistor-based+biosensors+with+a+digital+microfluidic+device+for+a+lab-on-a-chip+application.">Integration of field effect transistor-based biosensors with a digital microfluidic device for a lab-on-a-chip application.</a> Lab Chip. 12, 1533-1536 (2012).</li><li>Barbulovic-Nad, I., Yang, H., Park, P. S., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics+for+cell-based+assays.">Digital microfluidics for cell-based assays.</a> Lab Chip. 8, 519-526 (2008).</li><li>Gentilucci, L., de Marco, R., Cerisoli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modifications+designed+to+improve+peptide+stability:+incorporation+of+non-natural+amino+acids,+pseudo-peptide.">Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide.</a> 16, 3185-3203 (2010).</li><li>Miller, E. M., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+digital+microfluidic+approach+to+homogeneous+enzyme+assays.">A digital microfluidic approach to homogeneous enzyme assays.</a> Anal. Chem. 80, 1614-1619 (2008).</li><li>Jebrail, M. J., Bartsch, M. S., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics:+a+versatile+tool+for+applications+in+chemistry,+biology.">Digital microfluidics: a versatile tool for applications in chemistry, biology.</a> 12, 2452-2463 (2012).</li><li>Ng, A. H. C., Choi, K., Luoma, R. P., Robinson, J. M., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+Microfluidic+Magnetic+Separation+for+Particle-Based+Immunoassays.">Digital Microfluidic Magnetic Separation for Particle-Based Immunoassays.</a> Analytical Chemistry. 84, 8805-8812 (2012).</li><li>Sista, R. S., 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=Heterogeneous+immunoassays+using+magnetic+beads+on+a+digital+microfluidic+platform.">Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.</a> Lab Chip. 8, 2188-2196 (2008).</li><li>Malic, L., Veres, T., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochip+functionalization+using+electrowetting-on-dielectric+digital+microfluidics+for+surface+plasmon+resonance+imaging+detection+of+DNA+hybridization.">Biochip functionalization using electrowetting-on-dielectric digital microfluidics for surface plasmon resonance imaging detection of DNA hybridization.</a> Biosens. Bioelectron. 24, 2218-2224 (2009).</li><li>Malic, L., Veres, T., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+digital+microfluidics+for+enhanced+surface+plasmon+resonance+imaging.">Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging.</a> Biosens. Bioelectron. 26, 2053-2059 (2011).</li><li>Jebrail, M. J., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidic+method+for+protein+extraction+by+precipitation.">Digital microfluidic method for protein extraction by precipitation.</a> Anal. Chem. 81, 330-335 (2009).</li><li>Nelson, W. C., 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=Incubated+protein+reduction+and+digestion+on+an+electrowetting-on-dielectric+digital+microfluidic+chip+for+MALDI-MS.">Incubated protein reduction and digestion on an electrowetting-on-dielectric digital microfluidic chip for MALDI-MS.</a> Anal. Chem. 82, 9932-9937 (2010).</li><li>Mousa, N. A., 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=Droplet-scale+estrogen+assays+in+breast+tissue,+blood,+and+serum.">Droplet-scale estrogen assays in breast tissue, blood, and serum.</a> Sci. Transl. Med. 1, 1ra2 (2009).</li><li>Jebrail, M. J., 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=A+digital+microfluidic+method+for+dried+blood+spot+analysis.">A digital microfluidic method for dried blood spot analysis.</a> Lab Chip. 11, 3218-3224 (2011).</li><li>Choi, K., Ng, A. H. C., Fobel, R., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics.">Digital microfluidics.</a> Annual Review of Analytical Chemistry. 5, 413-440 (2012).</li><li>Jebrail, M. J., 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=Digital+Microfluidics+for+Automated+Proteomic+Processing.">Digital Microfluidics for Automated Proteomic Processing.</a> J. Vis. Exp. (33), e1603 (2009).</li><li>Thaitrong, N., 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=Quality+Control+of+Next+Generation+Sequencing+Library+through+an+Integrative+Digital+Microfluidic+Platform.">Quality Control of Next Generation Sequencing Library through an Integrative Digital Microfluidic Platform.</a> , (2012).</li><li>Shin, Y. J., Lee, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+vision+for+digital+microfluidics.">Machine vision for digital microfluidics.</a> Review of Scientific Instruments. 81, (2010).</li><li>Thornton, M., Eward, K. L., Helmstetter, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+minimally+disturbed+synchronous+cultures+of+hematopoietic+cells.">Production of minimally disturbed synchronous cultures of hematopoietic cells.</a> BioTechniques. 32, 1098-1105 (2002).</li><li>Huang, X., Dai, W., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforced+adhesion+of+hematopoietic+cells+to+culture+dish+induces+endomitosis+and+polyploidy.">Enforced adhesion of hematopoietic cells to culture dish induces endomitosis and polyploidy.</a> Cell Cycle. 4, 801-805 (2005).</li><li>Raje, M., 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=Charged+nylon+membrane+substrate+for+convenient+and+versatile+high+resolution+microscopic+analysis+of+Escherichia+coli+&amp;+mammalian+cells+in+suspension+culture.">Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli &amp; mammalian cells in suspension culture.</a> Cytotechnology. 51, 111-117 (2006).</li><li>Chatterjee, D., Hetayothin, B., Wheeler, A. R., King, D. J., Garrell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-based+microfluidics+with+nonaqueous+solvents+and+solutions.">Droplet-based microfluidics with nonaqueous solvents and solutions.</a> Lab Chip. 6, 199-206 (2006).</li><li>Perroud, T. 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=Microfluidic-based+cell+sorting+of+Francisella+tularensis+infected+macrophages+using+optical+forces.">Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces.</a> Analytical Chemistry. 80, 6365-6372 (2008).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+A+revolutionary+tool+for+transcriptomics.">RNA-Seq: A revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10, 57-63 (2009).</li><li>Malone, J. H., Oliver, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays,+deep+sequencing+and+the+true+measure+of+the+transcriptome.">Microarrays, deep sequencing and the true measure of the transcriptome.</a> BMC Biology. 9, (2011).</li><li>Wu, M., 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=Microfluidically-unified+cell+culture,+sample+preparation,+imaging+and+flow+cytometry+for+measurement+of+cell+signaling+pathways+with+single+cell+resolution.">Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.</a> Lab on a Chip. 12, 2823-2831 (2012).</li><li>Srivastava, N., 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=Fully+integrated+microfluidic+platform+enabling+automated+phosphoprofiling+of+macrophage+response.">Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.</a> Analytical Chemistry. 81, 3261-3269 (2009).</li><li>Herr, A. E., 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=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Bu mikro ölçekli hücre kültürü deneyleri ve uyarılması için basit ve çok yönlü bir otomatik bir platform geliştirdik. Kültür boyutları daha küçük çaplı mikrokapilerler kullanımı ile azaltılabilir; platformu bize küçük kültür hacimleri ve hücre popülasyonlarının (- - 20 ul ve 100 2.000 hücreleri, oda başına 1) ile çalışmasını sağlar. Bu ölçeklerde çalışan rutin çalışmaların maliyetini düşürür ve değerli reaktifler ve / veya hücrelerin kullanımı gerektire...

(In vitro analizinde) kültür muhafaza hücrelerin çalışma karmaşık biyolojik sistemlerin ve insan sağlığı düzenleyen temel ilke ve moleküler mekanizmaları içine değerli fikir sağlamıştır. Kültür, uyarılma ve Petri kutularına ve mikro-titer plakalar kullanmak analizi için hücreler, toplanması için konvansiyonel yöntemler mililitre çapta kültür hacmi hücreleri (≥ 10 5) (≥ 0.1 ml) arasında geniş popülasyonların çalışması için tasarlanmıştır. Ancak, hücrelerin sadece sınırlı bir miktarda mevcut olduğu bir çok durumda (örneğin, birincil hücreler), ya da küçük hücre popülasyonları (örneğin nüfus içerisindeki hücre-hücre değişkenliği azaltmak için) arzu edilir veya gerekli reaktiflerin elde etmek zordur veya pahalı (örneğin saflaştırılmış hücre-salgılanan faktörler). Bu tür sorunlar başarıyla kültür boyutunu küçültme tarafından ele alınabilir, hangi tüm tüketimi azaltmak yararı vardırReaktifler in vitro analiz 1,2 için gerekli. Ne yazık ki, geleneksel ekipman ve yöntemleri mikro ölçekli kültür ve 3-11 çok karmaşık ve en laboratuvarlar tarafından rutin kullanım için özel olan mevcut Mikroakiskan tabanlı otomatik sistemlerin hassas ve tekrarlanabilir manipülasyon desteklemez. Mikro ölçekli hacimlerde (1 - 20 ul) - Bu yazıda, otomatik kültür, stimülasyon ve hücrelerin küçük nüfus (2.000 hücre 100) geri kazanımı için basit ve çok yönlü teknoloji platformu montajı ve kullanımı açıklanmaktadır. Fibronektin kaplı mikrokapilerler (&quot;hücre perfüzyon odaları&quot; modülü) mikro ölçekli kültürlerin kurulması, bakım ve uyarılması için site olarak hizmet veren bir dizi ve bir dijital Mikroakiskan (DMF: platform mimarisi (Şekil 1) modüler yapıda olup &quot;transfer&quot; mikrokapilerler (&quot;merkez&quot; modülü) 14,15 yolları hücreleri ile donatılmış) 12,13 cihazve ve perfüzyon odaları reaktifleri. DMF kullanıcı ayrı ayrı aynı anda birden fazla damlacıkları ele ve cihaz donanım değiştirmeden manipülasyonlar (yani yeniden örnek işleme tren) değiştirmek veya yeniden sipariş edebilirsiniz. Onun büyük esneklik hücre kültürü 16,17, enzim testleri 18,19, immün 20,21, DNA analizi 22,23, protein işleme, 24,25 de dahil olmak üzere geniş bir uygulama yelpazesi, önemli bir teknoloji olarak son ortaya çıkması belirgindir ve klinik örnek işleme. 26,27 Bizim merkez DMF cihazlara doğal esneklik yararlanır, ve daha fazla uzman periferik manipülasyonlar bir alt kümesi (örneğin hücre kültürü) gerçekleştirmek için bir fırsat sunuyor microcapillary arayüzleri, ilavesi aracılığıyla artırır DMF yerine aygıtın kendisinde daha modülleri. Bu şekilde işleme tren bölümlere de pla tasarımı kolaylaştırırtform mimari (tüm işlem adımları taşıyabilen bir DMF cihaz oluşturmak için gerek yok) ve yeni fonksiyonlar olarak evrimi kolaylaştırır (sadece gerekli gibi yeni çevresel modüller entegre) gerekmektedir. Merkez içinde hücreleri ve reaktiflerin taşıma DMF cihaz 13,28 içinde elektrotlar sıralı aktivasyon tarafından oluşturulan kuvvetler electrowetting tarafından sürülür; gelen, ulaşım ve perfüzyon odaları içinde yüksek hassasiyetli şırınga tarafından oluşturulan basınç değişiklikleri tarafından desteklenmektedir pompa. Bu sıvı hareketlerin tüm basit bir elektronik arabirimi üzerinden kontrol ve önceden belirlenmiş komut kullanımı ile otomatik vardır. Temsili bir örnek olarak, bakteri ile meydan okuma (Şekil 2) üzerine bağışıklık hücrelerinde ortaya çıkardı transkripsiyonel yanıtların çalışma için platformun kullanımını göstermektedir. Platformda bu deneyler bize hücrelerin az sayıda (deneysel başına ~ 1000 ile çalışmak için etkintal durumu), deneme-to-deney değişkenliği, tasarrufu reaktifler ve emek eller yeniden doğrudan en aza indirmek. Bu avantaj sağladığını, hem de onun erişilebilirlik ve çok yönlülüğü göz önüne alındığında, bu platform laboratuar ve geniş bir uygulama çeşitli kullanım alanı bulur ve sadece sınırlı miktarlarda mevcut olan hücrelerin ve uyaranların çözümlenmesini kolaylaştıran çok özellikle faydalı olabilir gerekir.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

Kültür hücreleri (in vitro analizi) çalışması karmaşık biyolojik sistemlerin içine önemli fikir sağlamıştır. In vitro analizi için geleneksel yöntemler ve sistemler de mililitre ölçekli birimleri (≥ 0.1 ml) hücreleri (≥ 10 5), çok sayıda çalışma için uygundur. Ancak, bu ilgi ve / veya kültür, uyarılma ya da işlem için gerekli reaktiflerin hücre tüketimini azaltmak için kültür boyutunu aşağı ölçekte gerekli ya da arzu edilir olduğu birçok örneği vardır. Ne yazık ki, geleneksel yaklaşımlar mikro ölçekli kültürlerin hassas ve tekrarlanabilir manipülasyon desteklemez ve mevcut Mikroakiskan tabanlı otomatik sistemler çok karmaşık ve en laboratuvarlar tarafından rutin kullanım için uzmanlaşmıştır. Mikro ölçekli hacminde - Bu sorunu çözmek için, otomatik kültür, stimülasyon ve hücrelerin küçük nüfus (2.000 hücre 100) geri kazanımı için basit ve çok yönlü teknoloji platformu geliştirdiks (1-20 ul). Platform mikro ölçekli kültür, kurulan muhafaza ve uyarılır, içinde fibronektin kaplı mikrokapilerler (&quot;hücre perfüzyon odaları&quot;), bir dizi oluşur &quot;transfer&quot; mikrokapilerler (&quot;merkez ile donatılmış bir dijital Mikroakiskan (DMF) cihazı &quot;), ve perfüzyon odalarından yolları hücreleri ve reaktif olan, güçler perfüzyon odaları ve merkez arasında malzeme nakliyesi bir yüksek hassasiyetli şırınga pompası, hangi ve malzeme nakliyesi üzerinde kontrol sağlayan bir elektronik arayüz, hangi koordine ve önceden belirlenmiş komut ile otomatik. Bir örnek olarak, bakteri ile meydan bağlı bağışıklık hücrelerinde ortaya çıkardı transkripsiyonel yanıtlar çalışmayı kolaylaştırmak için platform kullanılır. Platformun kullanın bize, hücre ve reaktif tüketimini azaltmak deneme-için-deney değişkenliği en aza indirmek için etkin ve yeniden doğrudan eller emek. Bu bahşeder avantajları, hem de onun erişilebilirlik ve çok yönlülük, o göz önüne alındığındaur platformu laboratuar ve geniş bir uygulama çeşitli kullanım alanı bulur ve sadece sınırlı miktarlarda mevcut olan hücrelerin ve uyaranların çözümlenmesini kolaylaştıran çok özellikle faydalı olabilir gerekir.

Otomatik platformunun bir gösteri olarak, bu bakteri ile meydan bağışıklık hücrelerinin küçük nüfus mikro ölçekli Üreyen edildiği bir çalışma, yürütmek için kullanılan ve (Şekil 2 pro-inflamatuar yanıtların kapalı platform analizi için parçalanmış .) Altı hücre perfüzyonu odaların her büyüme ortamı 10 ml içinde tekrar süspanse 10 4 bağışıklık hücreleri (P388D.1 sıçangil makrofaj) ile tohumlandı. Bir bağlılık süre...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Biz mikro ölçekli hücre stimülasyonu deneyler için otomatik bir hücre kültürü ve sorgulama platformu geliştirdik. Platform yetiştirilmesi ve hücrelerin küçük nüfus uyarıcı ve moleküler analizler için lizatları kurtarma, basit, çok yönlü ve hassas kontrol sağlar. Platform de değerli hücreleri ve / veya reaktif kullanmak çalışmalar için uygundur.

Mikro ölçekli Hücre Stimülasyon deneyler için Çok Yönlü Otomatik Platformu

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K Görüntüleme.  Sandia National Laboratories. Biz mikro ölçekli hücre stimülasyonu deneyler için otomatik bir hücre kültürü ve sorgulama platformu geliştirdik. Platform yetiştirilmesi ve hücrelerin küçük nüfus uyarıcı ve moleküler analizler için lizatları kurtarma, basit, çok yönlü ve hassas kontrol sağlar. Platform de değerli hücreleri ve / veya reaktif kullanmak çalışmalar için uygundur.

Video: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotipik Kollajen Ben Assay: 3-Boyutlu Bağlamında Hücre Davranışı değerlendirin A Dövülebilir Platformu

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Biyomühendislik

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: Biyokütle Enzim Discovery ve Kokteyl Optimizasyonu Otomatik Platform

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Sağlam, mikroakışkan Hücre içi Delivery Platform olarak hücre Sıkma

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Hücre ve Doku mechanobiology Applications A Novel Germe Platformu

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Yüksek verim Tek hücreli İzolasyonu ve Kültür mikroakışkan Platformu

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Katmanlı Aljinat Yapıları: Heterojen hücre popülasyonlarının Co-kültür için bir platform

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Hücre farklılaşması ve Çekiş Kuvvetleri Korelatif Analizi için Yüksek verim Hücre Mikroarray Platformu

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Desenlendirme Proteinlerin ve Hücreleri Çok Yönlü Yöntemi

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA nanotüpler Semiflexible polimerler eğitim için çok yönlü bir araç olarak

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Uzun Süreli Hücresiz Gen Ekspresyonunun İletimi için Çok Katmanlı Mikroakışkan Platform

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...