Bu çalışma Ulusal Sağlık ve Tıp Araştırma Konseyi Avustralya (proje hibe hayır °. 6.288.480) ve Scobie ve Clare MacKinnon Güven tarafından desteklenmiştir.

1. RT Reaksiyon Micromixing <ol><li> Micromixing RT tepki gerçekleştirmeden önce, RT tepki istenilen sıcaklığa micromixer dengelenmesi. <ol><li> Micromixer 37 içine yerleştirin ° C (veya RT tedarikçi tarafından tavsiye edilen sıcaklık) inkübatör RT reaksiyonun başlangıcı için en az 1 saat önce için. </li></ol></li><li> RT karışımı ters transkriptaz tedarikçisi göre ayarlayın (örneğin Promega, Qiagen gelen Omniscript MMTV-RT) talimatları yerine ng-mg giriş toplam RNA (örneğin 0.1-1pg/μl), tek hücreli miktarda hariç tedarikçiler tarafından tavsiye edilen miktarda. <ol><li> Biz standart steril, nükleaz 200μL ince duvarlı PCR tüpleri kullanırlar. </li></ol></li><li> Karıştırma için hazır olan en kısa sürede güvenli bir şekilde micromixer içine pozisyon RT tüpler. Micromixer RT reaksiyon süresi 37 ° C inkübatör içinde kalmalıdır. </li><li> Uygun frekans ve micromixing genlik seçin. Bu örnekte, 150Hz kullanın. </li><li> Micromixer açın ve RT tepki (60 dakika) süresi boyunca bekletin. </li><li> Micromixer kapatın ve RT reaksiyon tamamlandıktan sonra RT tüpleri almak. </li><li> RT tüpleri, buz üzerinde yerleştirin ve kantitatif Polimeraz Zincir Reaksiyonu (qPCR veya gerçek-zamanlı PCR) gibi daha fazla uygulamaları ile normal olarak devam edin. </li></ol> 2. Temsilcisi Sonuçlar: RT reaksiyonlar bağlamında micromixing yararı cDNA verimi diğer karıştırma yöntemleri (örneğin, sallayarak karıştırın veya öğütme) kullanıldığında göreceli bir artış. Bu cDNA kullanarak kantitatif Polimeraz Zincir Reaksiyonu (qPCR veya gerçek-zamanlı PCR) sonraki analizi üzerine, örneğin, görülebilmektedir. Şekil 2A hemen önce RT inkübasyon (siyah, &quot;öğütme&quot; RT reaksiyon öğütme ile karşılaştırıldığında, ilk 5 dakika için micromixing etkileri (mavi, &quot;micromixed5&quot;) ya da tüm 60 dakika (kırmızı, &quot;micromixed60&quot;) göstermektedir .) Bu durumda önceki RT reaksiyon hacmi 25μL ve total RNA 2.5pg içeriyordu. cDNA temsil Nurr-1 mRNA algılamak için tasarlanmış primerler kullanılarak qPCR izleri yukarıda gösterildiği anlamına gelir ± 3 Bu aynı deneyi tekrarlar% 50 maksimum floresans ulaşmak için döngüleri sayısı SEM&#39;ler aşağıda çizilir. Yıldız işareti (Tukey çoklu karşılaştırmalar tek yönlü ANOVA) istatistiksel olarak anlamlı bir farklılık göstermektedir. Önceki RT reaksiyon tüm 60 dakika boyunca micromixing öğütme üzerinde önemli bir gelişme ise önceki RT reaksiyon ilk 5 dakika micromixing öğütme üzerinde anlamlı olmayan iyileşme üretilen, büyüklüğü yaklaşık 15 qPCR eşdeğer döngüleri, ortalama. Micromixing yararı da elde edilen sonraki qPCR ürünlerin erime eğrisi analizinin görülebilir. Sadece tutarsız qPCR üretilen ilk 5 dakika veya öğütme için micromixing ise, önceki RT reaksiyon tüm 60 dakika boyunca micromixing Şekil 2B gösterilen deney, tutarlı bir yinelenen örnekleri qPCR sinyal (Şekil 2BA, 2 kırmızı izleri) sonuçlandı izleri (Şekil 2BA, 2 mavi izleri ve 2 siyah izleri, sırasıyla). Gri izleri qPCR reaksiyon kaldı cDNA hangi negatif kontroller (&quot;negatif&quot;). Bu deneyde qPCR ürünleri belliydi sıcaklığı (yani erime eğrisi analizi) Ramping denatüre olduğunu ne zaman uygun PCR ürünleri (yani, mRNA çok daha yüksek konsantrasyonlarda ters qPCR transkripsiyonu ve amplifiye edildi, elde edilen aynıydı bir Amplikon gösterilmemiştir) 60 dakika (Şekil 2BB, 2 kırmızı izleri) micromixed olmuştu sadece RT reaksiyonlar sunuldu. Tüm diğerleri (yani micromixed5, öğütme ve negatif kontroller), alternatif Amplikon ürünler (örneğin, astar dimerleri) oluşturulur. <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Şekil 1"/> Şekil 1 standart laboratuvar RT reaksiyonlara micromixing uygulamak için protokol Akış diyagramı. Referanslar 13,14 fiziksel prensibi yer micromixer ve nasıl bir in-house micromixer inşa etmek için daha ayrıntılı açıklamalar verilmiştir <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Şekil 2"/> Şekil 2 RT aksi standart laboratuvar RT reaksiyonlarda mRNA tek hücreli miktarlarda oluşan reaksiyonlar önceki uygulanan micromixing faydaları gösteren Temsilcisi qPCR veri Nurr-1 genini kodlayan cDNA temsil eden mRNA tespit qPCR izleri Bir ​​Örnek. Önceki RT reaksiyonlar 25μL hacmi toplam RNA 2.5pg yer alan ve 37 ° C&#39;de 60 dakika için bir kuluçka yapıldı. RT reaksiyonlar R öğütme tarafından karıştırıldıT RT reaksiyon (mavi izleri &quot;micromixed5&quot;) ilk 5 dakika boyunca (kırmızı izleri &quot;micromixed60&quot; micromixed veya RT reaksiyon tüm 60 dakika boyunca micromixed inkübasyon (siyah izleri &quot;öğütme&quot;) önce karışmaktadır .) Ortalamalar ±% 50 maksimum floresans ulaşmak için gerekli qPCR döngülerinin sayısı SEM&#39;ler aşağıda çizilir n = 3 bu deneyi tekrarlar. Asterisk (Tukey çoklu karşılaştırmalar tek yönlü ANOVA) anlamlı bir farklılık gösterir. B bir başka qPCR deney 25μL hacmi toplam RNA içeren 25pg RT reaksiyonlar aşağıdaki Nurr-1 cDNA ürünü inceleyerek, bu sefer. qPCR izlerinin yanı sıra elde edilen qPCR ürün eğrisi analizi (a) erime (b) farklı karıştırma koşullar için gösterilmiştir. Hiçbir cDNA önceki RT tepki olarak dahil edildiği negatif kontrolleri de (gri izleri, &quot;negatif&quot;) yer almaktadır. MRNA çok daha yüksek konsantrasyonlarda RT reaksiyonlar da yapılan ve (b), micromixed60 için (kırmızı izlerini) örnekleri aynı erime eğrisi zirveleri (veriler gösterilmemiştir) üretildi .

<ol>
	<li>Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Funct+Genomic+Proteomic.">Brief Funct Genomic Proteomic.</a> 2 (1), 31-31 (2003).</li><li>Aumann, T. D., Gantois, I., Egan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exp Neurol. 213 (2), 419-419 (2008).</li><li>Ottino, J. M., Wiggins, 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> Philos Transact A Math Phys Eng Sci. 362 (1818), 923-923 (2004).</li><li>Losey, M. W., Jackman, R. J., Firebaugh, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Microelectromech. Syst. 11, 709-709 (2002).</li><li>Bontoux, N., Dauphinot, L., Vitalis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab Chip. 8 (3), 443-443 (2008).</li><li>Marcus, J. S., Anderson, W. F., 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=.">.</a> Anal Chem. 78 (9), 3084-3084 (2006).</li><li>Marcus, J. S., Anderson, W. F., 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=.">.</a> Anal Chem. 78 (3), 956-956 (2006).</li><li>Warren, L., Bryder, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc Natl Acad Sci U S A. 103 (47), 17807-17807 (2006).</li><li>Jiao, Z. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 6, 847-847 (2009).</li><li>Ahmed, D., Xiaole, M., Juluri, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 7, 727-727 (2009).</li><li>Paxton, W. F., O'Hara, M. J., Peper, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 80, 4070-4070 (2008).</li><li>Autom, L. a. b. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 11, 399-399 (2006).</li><li>Petkovic-Duran, K., Manasseh, R., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 47 (4), 827-827 (2009).</li><li>Boon, W. C., Petkovic-Duran, K., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 50 (2), 116-116 (2011).</li><li>Liu, R. H., Lenigk, R., Druyor-Sanchez, 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=.">.</a> Anal Chem. 75 (8), 1911-1911 (1911).</li><li>Riley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ann+Rev+Fluid+Mech.">Ann Rev Fluid Mech.</a> 33, 43-43 (2001).</li><li>Whitehill, J., Neild, A., Ng, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied Physics Letters. 96 (5), 053501-053501 (2010).</li></ol>

Yazarlar ifşa etmek için bir şey var.

Standart laboratuvar RT reaksiyonlara micromixing uygulama yöntemi, tabii ki, tutabilen burada açıklanan herhangi bir yöntemle (örneğin, hücre parçalama, lazer yakalama mikrodiseksiyon) ile hasat mRNA. Aynı zamanda herhangi bir marka veya türleri RT reaktifleri, herhangi bir sıcaklık (micromixer malzemelerin tolerans içinde), ve herhangi bir süre oluşur. Örneğin, RT rastgele hexamer veya oligo-dT primerleri oluşan reaksiyonlar gelişmiş cDNA verim gözlemledik. Aşağıdaki kısıtlamalara uygun diğe...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Total RNA ek bileşenini dondurulmuş akut hazırlanan yetişkin farenin orta beyin dilimleri izole edildi </td><td></td><td></td><td></td></tr><tr><td> PicoPure RNA İzolasyon Kiti </td><td> Arcturus, CA, USA </td><td> KIT0204 </td><td> Kiti artık Applied Biosystems edinilebilir. </td></tr><tr><td> DNA ücretsiz DNaz Arıtma ve Kaldırma Reaktifler </td><td> Ambion </td><td> AM1906M </td><td></td></tr><tr><td> Rastgele hexamer primerleri </td><td> Promega </td><td> C1181 </td><td></td></tr><tr><td> AMV-RT </td><td> Promega </td><td> M5101 </td><td></td></tr><tr><td> dNTP seti </td><td> Promega </td><td> U1240 </td><td></td></tr><tr><td> RNasin Ribonükleaz İnhibitörü </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Nükleazlar-Ücretsiz Su </td><td> Promega </td><td> P1193 </td><td></td></tr><tr><td> SYBR Yeşil PCR Master Mix </td><td> Uygulamalı Biosystem </td><td> 4309155 </td><td></td></tr><tr><td> Hprt ileri (20mer): CTT TGC TGA SKK GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> Hprt ters (20mer): TAT GTC CCC CGT TGA CTG AT </td><td></td><td></td><td></td></tr><tr><td> Nurr1 ileri (23mer): CAG CTC CGA TTT CTT AAC TTK AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 ters (23mer): GGT GAG GTC CAT GCT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> 1000 NanoDrop Spektrofotometre. </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Corbett Rotor Gen RG-6000 </td><td> Corbett Yaşam Bilimleri </td><td></td><td> Corbett Yaşam Bilimleri QIAGEN tarafından Temmuz 2008&#39;de satın alındı </td></tr></tbody></table>

increasing cdna yields from single-cell quantities of mrna in standard laboratory reverse transcriptase reactions using acoustic microstreaming

Gen ekspresyonu hücre davranışı ile ilişkilendirerek ideal tek hücre düzeyinde yapılır. Ancak, tek bir hücrenin (1-5, 1-50pg toplam RNA ya da% 0.01-2.5pg mRNA, hücre başına 1) mevcut labil mRNA transkripsiyonu tersine çevirmek için önce küçük bir miktar daha çok düşürür, çünkü bu kolayca elde değil . istikrarlı bir cDNA kopyası haline. Örneğin, standart laboratuar reaktifleri ve donanım kullanarak, genlerin sadece az sayıda hücre başına 2 nitel değerlendirilir olabilir. MRNA tek hücreli miktarda oluşan standart laboratuvar ters transkriptaz (RT) reaksiyonlar (mikrolitrelik hacimleri yani standart reaktifler) verimliliği artırmak için bir şekilde düşürür önce mRNA cDNA dönüştürülebilir, böylece daha hızlı reaktifler karıştırmak olacaktır. Ancak mikrolitrelik ölçekler sıvı akışı laminer olduğunu, çünkü bu önemsiz değildir, yani mevcut karıştırma yöntemleri (sallayarak, vorteks ve öğütme yani), etkin bir şekilde reaktifleri karıştırmak için yeterli kaotik hareket üretemez. Bu sorunu çözmek için, mikro ölçekli karıştırma teknikleri 3,4 kullanılır. Mikroakışkan tabanlı karıştırma teknolojileri başarıyla RT reaksiyon verimi 5-8 artış geliştirilmiştir. Ancak, Mikroakiskan teknolojileri, nispeten pahalı ve henüz yaygın değil özel bir donanım gerektirir. Daha ucuz, daha rahat bir çözüm arzu edilir. Bu çalışmanın ana amacı, mRNA tek hücreli miktarlarda oluşan standart laboratuvar RT reaksiyonlar bir romanı &quot;micromixing&quot; tekniği uygulaması cDNA verimi önemli ölçüde artırır nasıl göstermektir. Biz cDNA verim sağlar yaklaşık 10-100-katlama, artış bulabilirsiniz: (1) daha fazla sayıda hücre başına analiz edilmesi gereken genler, gen ekspresyonu (2) daha düşük bolluk genlerin kantitatif analiz ve (3) daha iyi algılama tek hücre. Micromixing akustik, 9-12 küçük bir engel etrafında yayılan ses dalgalarını engel yakın bir ortalama akışı oluşturmak bir fenomen microstreaming dayanmaktadır. Biz kilit bir sadeleştirme ile akustik bir microstreaming tabanlı cihaz (&quot;micromixer&quot;); akustik microstreaming sistemi, küçük bir eğrilik 13 yarıçapı ile bir sıvı-hava arayüzü sağlayarak ses frekansları elde edilebilir . Bir tüp bir çözüm mikrolitrelik hacmi menisküs uygun küçük bir eğrilik yarıçapı sağlar. Ses frekanslarının kullanımı, donanım ucuz ve 13 çok yönlü olması anlamına gelir ve standart laboratuvar sonicators olabileceği gibi, nükleik asitler ve diğer biyokimyasal reaktifler zarar görmezler.

Watch this Scientific Journal Video about Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming at JoVE.com

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Biz aksi takdirde standart laboratuvar ters transkripsiyon reaksiyonları mRNA tek hücreli miktarda cDNA verimi artırmak için yeni bir yöntem açıklanmaktadır. Yenilik, titreme, vorteks veya öğütme daha etkili mikrolitrelik ölçekler sıvıları karıştırmak için, akustik microstreaming fenomeni kullanan bir micromixer, kullanımı bulunur.

Akustik Microstreaming kullanarak Standart Laboratuvar Reverse Transcriptase reaksiyonlarda mRNA Tek hücreli Miktarları cDNA Verim artırılması

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

increasing-cdna-yields-from-single-cell-quantities-mrna-standard

Research

JoVE Journal

Bioengineering

10.1K Görüntüleme.  University of Melbourne. Biz aksi takdirde standart laboratuvar ters transkripsiyon reaksiyonları mRNA tek hücreli miktarda cDNA verimi artırmak için yeni bir yöntem açıklanmaktadır. Yenilik, titreme, vorteks veya öğütme daha etkili mikrolitrelik ölçekler sıvıları karıştırmak için, akustik microstreaming fenomeni kullanan bir micromixer, kullanımı bulunur.

Video: Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Standart Mürekkep Püskürtmeli Yazıcı Kullanımı Geçici Hücre Zarı Gözenekler oluşturma

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Biyomühendislik

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Tek Hücre Salgıları uzay-zamansal Görüntüleme için bir etiket içermeyen Tekniği

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Tek hücreli genom sıralama için bir üretilen iş Ultrahigh mikrosıvısal Platform

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

Referansta bulunmadığı Pasifik Istiridyeleri neredeyse sıralı cDNA Kütüphanesi üretimi için bir converging stratejisi

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

Biyolojik Olarak Aktif Mercan Matrisi Kullanılarak Ayrışmış Nöral Hücre Kültürlerinin Dayanıklılığının Arttırılması

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Akustik Ayırıcı Kullanarak CHO Hasat Edilen Hücre Kültürü Sıvısının Birincil Netleştirilmesi

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

Ahşap Yapıştırıcıların Yapışkan Mukavemet Gelişimi için Standart Test Yöntemi ASTM D 7998-19

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Manyetik Hipertermi ve Pothototermal Kombinasyon Kanser Tedavisi için Manyetik, Akustik ve Optik-Üçlü Duyarlı Mikrobubbles

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

İnsan kaynaklı Pluripotent Kök Hücre Türevli Kardiyomiyositlerde Tek Hücreli Optik Eylem Potansiyeli Ölçümü

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Mikrodesenli Elastomerler Kullanarak Tek Hücreli Kasılmanın Basitleştirilmiş, Yüksek Verimli Analizi

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...