Bu proje, MIT&#39;de Deshpande Teknolojik Yenilik Merkezi (RK ve JMK) ve Kimyasal ve Biyolojik Ayrımları programı ile RK NSF KARİYER ödül 0.952.493 tarafından desteklenmiştir. Biz Asker Nanoteknolojiler (ISN) ve tesislerin kullanımı için MIT&#39;de Microsystems Teknoloji Laboratuvarı (MTL) Institute for teşekkür ederim.

1. Haddeleme HL60 hücre 1.1. Desenli Yüzeyler imalatı. <ol><li> Altın kaplamalı cam slaytlar PEG moleküllerinin kendi kendini monte mono tabakaları (SAMs) alternatif yapmak microcontact baskı (μCP) 4-7 kullanma: α = 10, eğim açısı ile reseptör desenleri tanımlanan microcontact baskı polidimetilsiloksan (PDMS) pulları Üretiyor SU-8 kalıplama işlemi °. 20 dakika boyunca altın piranha çözüm (sülfürik asit 03:01 karışımı% 30 hidrojen peroksit) ile yüzeyi temizleyin ve sonra, bol DI su ile 24.5 ° C önceden az yüzey durulayın. Etanol 5mM PEG solüsyonu ile Mürekkep PDMS damgası. 20 dakika boyunca hava damga kurulayın. 40 saniye süreyle yavaşça altın yüzeyinde pul koymak ve altın yüzey arasında iyi bir iletişim ve damgası olduğundan emin olun. Aşırı basınç gerek yoktur. Etanol ile yüzey N 2 akışı altında durulayın ve kurulayın. </li><li> Yüzey 24,5 az 3 saat boyunca perfüzyon odası (Elektron Mikroskobu Bilimleri) kullanarak ° C desen P-selektin ile diğer alanlar. P-selektin çözüm (15 mg / DPBS mL) içinde inkübe Yüzey bol DPBS ile durulayın. </li><li> Dolgu BSA (DPBS 1 mg / ml) ile yüzey 1 saat non-spesifik etkileşimleri engellemek için. Yüzey bol DPBS ile durulayın. </li></ol> 1.2. Hücre Akış Odası Rolling Deneyler. <ol><li> HL60 hücreleri (~ 10 5 hücre / ml) (genişliği w = 1,0 cm, uzunluğu = 6 cm; Glycotech, Inc yüksekliği h = 0,0127 cm) dikdörtgen bir akış odasında desenli yüzeyler üzerinde bir süspansiyon Akış 24.5 ° C 75 mcL / dak akış hızı üretmek için yaklaşık 0.5 dyn / cm 2 (~ 0.05 Pa) karşılık gelen kayma gerilmesi, bir şırınga pompası (Dünya Hassas Aletler, SP230IW) kullanın . Kayma gerilmesi τ uçağın Poiseuille hacimsel debisi Q μ kinematik viskozitesi (0.001002 Pa ler) τ = 6μQ/wh 2, akış denklemi kullanarak hesaplayın, w akışını odasının genişliği ve h yüksekliği akış odasına. </li><li> 300 süreleri için genellikle saniyede 1 kare hızında, 4 kullanarak yapışkan P-selektin yüzeyler ile etkileşim haddeleme HL60 hücreleri kaydetmek için monte edilmiş bir kamera (Andor IXON 885) × amacı ile bir inverted mikroskop (Nikon TE2000-U) kullanın s Her kayma gerilmesi büyüklüğü ve desen eğim açısı için üç bağımsız deneyler, gerçekleştirin. Her deneyden elde edilen ortalama değerler ortalama ve standart sapma gibi mevcut veri. </li><li> Veri Analizi. Görüntü dizileri bir parçacık desenli hat kenarlarında parçaları üretmek için ücretsiz 8 izleme kullanılan özelleştirilmiş bir Matlab (Mathworks, Inc.) Programı tarafından analiz edin. P-selektin bandının sonuna kadar uzanan parçalar seçilir ve iki düz çizgi parçaları ile donatılmış akışı ile uyumlu bir desen kenarı ile uyumlu diğer. Bu iki parça daha sonra kenarına hız haddeleme, ve düz bir bölgeye hız haddeleme, kenar izleme uzunluğunu hesaplamak için kullanılır. </li></ol> 2. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/2640/2640fig1.jpg" alt="Şekil 1" /> Şekil (A) 4 × amacı kullanarak yapışkan P-selektin yüzeylerde HL60 haddeleme etkileşimleri video dönüştürülür mikroskop görüntülerini bir gösterir. Parlak ve koyu bölgeler sırasıyla, P-selektin reseptör ve PEG bölgelere karşılık gelir. Şekil (B) özelleştirilmiş bir Matlab programı kullanılarak elde parçalarını gösterir. Kenar eğim açısı 10 ° ve kayma gerilmesi 0.5 dyn / cm 2 idi. (C-2), kenar izleme uzunluğu, l e, deplasman, d ve kenarında ve haddeleme hızları sırasıyla bantlar, V e ve V p, içinde, (C-1) Şekil olarak açıklanmıştır. Şekil gösterir kenar izleme uzunluğu dağılımı (kaydedilen hücrelerinin sayısı). Insets l e ortalama değer ve kenarında haddeleme hızı (V e) ve (V p), eğim açısı, bantları içinde α = 10 ° ve sıvı shear stresin büyüklüğü 0.5 dyn / cm 2 civarında . Hata çubukları, bir standart sapma, n = 3, her durum için çoğaltmak denemeleri (3 çoğaltmak yüzeyler) temsil

<ol>
	<li>Karnik, R., Hong, S., Zhang, H., Mei, Y., Anderson, D. G., Karp, J. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+control+of+cell+rolling+in+two+dimensions+through+surface+Patterning+of+receptors.">Nanomechanical control of cell rolling in two dimensions through surface Patterning of receptors.</a> Nano Lett. 8 (4), 1153-1158 (2008).</li><li>Lee, C. H., Bose, S., Van Vliet, K. J., Karp, J. M., Karnik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+Lateral+Displacement+of+HL60+Cells+Rolling+on+Asymmetric+P-selectin+Patterns.">Examining Lateral Displacement of HL60 Cells Rolling on Asymmetric P-selectin Patterns.</a> Langmuir. 27 (1), 240-249 (2011).</li><li>Norman, K. E., Moore, K. L., McEver, R. P., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+rolling+in-vivo+is+mediated+by+p-selectin+glycoprotein+ligand-1.">Leukocyte rolling in-vivo is mediated by p-selectin glycoprotein ligand-1.</a> Blood. 86 (12), 4417-4421 (1995).</li><li>Bernard, A., Delamarche, E., Schmid, H., Michel, B., Bosshard, H. R., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+patterns+of+proteins.">Printing patterns of proteins.</a> Langmuir. 14 (9), 2225-2229 (1998).</li><li>James, C. D., Davis, R. C., Kam, L., Craighead, H. G., Isaacson, M., Turner, J. N., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+protein+layers+on+solid+substrates+by+thin+stamp+microcontact+printing.">Patterned protein layers on solid substrates by thin stamp microcontact printing.</a> Langmuir. 14 (4), 741-744 (1998).</li><li>Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+self-assembled+monolayers+to+understand+the+interactions+of+man-made+surfaces+with+proteins+and+cells.">Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells.</a> Annual Review of Biophysics and Biomolecular Structure. 25, 55-78 (1996).</li><li>Tan, J. L., Tien, J., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+of+proteins+on+mixed+self-assembled+monolayers.">Microcontact printing of proteins on mixed self-assembled monolayers.</a> Langmuir. 18 (2), 519-523 (2002).</li><li>Lee, D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+Printing+of+P-Selectin+Increases+the+Rate+of+Neutrophil+Recruitment+Under+Shear+Flow.">Microcontact Printing of P-Selectin Increases the Rate of Neutrophil Recruitment Under Shear Flow.</a> Biotechnology Progress. 24 (5), 1052-1059 .</li><li>Greenberg, A. W., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+mediated+by+differential+rolling+adhesion.">Cell separation mediated by differential rolling adhesion.</a> Biotechnol. Bioeng. 73 (2), 111-124 (2001).</li><li>Higuchi, A., Tsukamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+of+hepatocytes+and+fibroblasts+through+surface-modified+polyurethane+membranes.">Cell separation of hepatocytes and fibroblasts through surface-modified polyurethane membranes.</a> J. Biomed. Mater. Res. Part A. 71A (3), 470-479 (2004).</li><li>Alexeev, A., Verberg, R., Balazs, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+surfaces+segregate+compliant+microcapsules.">Patterned surfaces segregate compliant microcapsules.</a> Langmuir. 23 (3), 983-987 (2007).</li></ol>

Biz microcontact baskı 2 kullanılarak üretilen asimetrik reseptör desenli yüzeyler hücre haddeleme yörüngeleri incelemek için bir protokol tanımlanmıştır. PEG ve P-selektin alanlar arasında belirgin kontrast gösteren desenli yüzey optik mikroskop görüntüleri damgalama başarılı olup olmadığını teyit etmek için kullanılabilir. Keskin, düz kenarlar damgalama iyi yapıldığında görülebilir. Hard damga basarak desen hassas sınırlar damga deformasyona neden olabilir. Dalgalı kenarları mürekkep konsantrasyonu çok yüksek veya damgalama süresi çok uzun zaman elde edilebilir. Damga boyutu çok küçük (&lt;0.5 cm 2) zaman damgası ve yüzey arasındaki Kötü temas üniform olmayan PEG desen yol açar. Sigara taze mürekkep bir çözüm, çünkü P-selektin etkisizleştirmek için azalmış kabiliyet PEG desenli bölgeleri üzerinde yuvarlanan hücrelerinde neden olabilir. Gerçek eğim açısı, yüzeyler ile etkileşim değil, serbest akan hücrelerinin desen ve yörüngeleri görüntüleri hesaplanabilir. Deney desen kenarlarında yuvarlanma olaylar bireysel hücre hakkında bilgi verim açıklanan, ancak bunu otomatik olarak bir desen ayırmak ve başka bir desen üzerinde yeniden takmak hücreleri izlemek için zordur. Desen aralıkları değiştirme, örneğin, tek bir hücrenin birden fazla kenarları karşılaşmadan izlemeyi etkinleştirmek olabilir. Bu yetenek, tek hücre düzeyinde farklı haddeleme özelliklere sahip hücreler arasındaki ayrım daha yüksek çözünürlük sağlar. Haddeleme davranış hücre hücre ligandlar bağlı olarak, bu tür deneyler uygun desen tasarımı, 9-12 3, haddeleme davranış farklılıkları dayalı farklı hücre fenotipleri ayrı etkinleştirebilirsiniz hipotez . Bu çalışma, bu nedenle cihazların tasarımı için hücre ligandlar ve desenli asimetrik reseptörleri arasındaki etkileşim dayalı hücre ayırma ve analizi için yararlı olabilir.

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Human promyelocytic leukemia cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CCL-240</td>
<td>HL60 cells</td>
</tr>
<tr>
<td>Gold-coated glass slides</td>
<td>&nbsp;</td>
<td>EMF</td>
<td>TA134</td>
<td>Gold slides</td>
</tr>
<tr>
<td>(1-Mercaptoundec-11-yl)tetra(ethylene glycol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>674508</td>
<td>PEG</td>
</tr>
<tr>
<td>Recombinant human P-selectin</td>
<td>&nbsp;</td>
<td>R&amp;D Systems Inc.</td>
<td>ADP3-050</td>
<td>P-selectin</td>
</tr>
<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals, Inc.</td>
<td>BSA-50</td>
<td>BSA</td>
</tr>
<tr>
<td>Dulbecco's phosphate buffered saline </td>
<td>&nbsp;</td>
<td>Mediatech Inc.</td>
<td>21-030</td>
<td>DPBS</td>
</tr>
<tr>
<td>Sulfuric acid </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>339741</td>
<td>&nbsp;</td>
<tr>
<td>Hydrogen peroxide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>316989</td>
<td>&nbsp;</td>		</tbody>
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studying cell rolling trajectories on asymmetric receptor patterns

Yanal deplasman asimetrik reseptör desenleri üzerinde yuvarlanan bir akış akışa dik hücreleri, hücreler 1 etiket ayırma ve analiz için yeni cihazlar geliştirilmesi için bir fırsat sunar . Bu tür cihazlar sürekli akış ayrılması, ya da farklı hücre fenotipleri veya reseptör ekspresyonu düzeyleri birbirinden ayırt etmek yapışma modüle reseptör desen yanal deplasman kullanabilirsiniz. Hücre reseptör-desenli yüzeyler üzerinde yuvarlanma yörüngeleri doğasını anlamak yüzeyleri ve bu tür cihazların tasarım mühendisliği için gerekli. Burada, biz, hücre haddeleme yapışma 2 destekleyen asimetrik reseptör desen hücre haddeleme yörüngeleri eğitim için bir protokol göstermektedir . İyi tanımlanmış, P-selektin reseptörleri mikron çaplı bir desen, bir akış odasında dahil edildi altın kaplı slaytlar üzerinde baskı microcontact kullanılarak imal edildi. PSGL-1 ligand 3 ifade HL60 hücreleri desenli çizgiler bir alan boyunca aktı ve ters bir aydınlık alan mikroskobu görüntülendi. Hücreler lateral çökme 1 desen eğimli kenarları boyunca rulo ve paletli. Her hücre, tipik olarak belirli bir mesafe (kenar izleme uzunluğu olarak tanımlanır) desen kenarlarında haddelenmiş kenarına kopuk ve bir alt-desen yatışma. Bu dekolmanı zor akışını odasına çıkmak için girişten itibaren bir hücrenin tüm yörünge takip hale getirse de, parçacık izleme yazılımı, tek bir reseptör üzerinde hareket olduğu zaman yuvarlanma sırasında hücrelerin yörüngeleri analiz ve verim desenli hat. Yörüngeleri, daha sonra hücre haddeleme hızları ve her hücre için farklı desen izleme kenar uzunlukları dağılımları elde etmek için incelendi. Bu protokol, hücre reseptör desen haddeleme yörüngeleri ölçülmekte ve desen açısı ve kayma gerilmesi gibi bu mühendislik parametrelerini ilgili yararlıdır. Bu tür veriler, etiket hücre ayırma ve analiz için mikroakışkan cihazların tasarımı için faydalı olacaktır.

Watch this Scientific Journal Video about Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns at JoVE.com

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Biz asimetrik reseptör-desenli yüzeyler üzerinde hücre haddeleme yörüngelerini gözlemlemek ve analiz etmek için bir protokol açıklar. Ortaya çıkan veriler etiket ücretsiz cep ayırma ve analiz için reseptör-desenli yüzeylerin mühendisliği için yararlıdır.

Asimetrik Reseptör Desenler Hücre Rolling Yörüngeler incelenmesi

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

studying-cell-rolling-trajectories-on-asymmetric-receptor-patterns

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K Görüntüleme.  MIT - Massachusetts Institute of Technology. Biz asimetrik reseptör-desenli yüzeyler üzerinde hücre haddeleme yörüngelerini gözlemlemek ve analiz etmek için bir protokol açıklar. Ortaya çıkan veriler etiket ücretsiz cep ayırma ve analiz için reseptör-desenli yüzeylerin mühendisliği için yararlıdır.

Video: Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Hücre-malzeme Etkileşim Araştırmalar Nanogözenekli Altın Patterns mikroimalat

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Biyomühendislik

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

Sistematik Analizi<em&gt; In Vitro</emÇok-çukurlu plaka mikroakışkan Sistemi ile&gt; Hücre Rolling

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Photolithographically Tanımlı Parylene-C hücre Desenlendirme: SiO<sub&gt; 2</sub&gt; Yüzeyleri

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

Protein Analiz Sürekli Tanıma Desenler oluşturma Elektronik Dil

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Floresan biyomembran Kuvvet Probe: Tek Hücre üzerinde Reseptör-ligand Kinetiklerinin Eşzamanlı kantitasyonu ve Bağlama kaynaklı Hücre içi Sinyal

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Katı Lipit nanopartiküller (SLNs) Hücre içi Hedefleme için başvurular

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Korelatif Mikroskopisi kullanılarak sağlam hücrelerde Epidermal Büyüme Faktörü Alıcısının stokiyometrisinden incelenmesi

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Beyin kanseri ve nörodejeneratif hastalıkların Incelenmesi için insan nöral Organoidler

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

Polimerik Yüzeylerde Adsorbe Protein Katmanlarında Sinyalizasyonu NF-kB/AP-1 Sinyallerini İnceleyecek Bir Makrofaj Muhabiri Hücre Tetkik

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Bir İnsan 3D Ekstrasellüler Matrix-Adiposit Kültür Modeli Matrix-Hücre Metabolik Crosstalk Çalışma için

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...