Name: adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface
Uploaded: 2011-11-02T13:00:00
Description: Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Bu çalışma NIH hibe programı R01HL091020 R01HL093723, R01AI077343 ve R01GM096187 tarafından desteklenmiştir.

1. Tam kan eritrosit izolasyonu <ol><li> EAS-45 çözümleri hazırlayın. Tablo I tüm malzemeyi tartılır ve 100-200ml DI su içinde çözülür. 1000ml çözüm yapmak ve 8.0 pH ayarlamak için su ekleyin. 50ml Filtre ve kısım. Saklama için -20 ° C&#39;de dondurun. </li></ol> Not: Adım 1.2 Kurumsal Değerlendirme Kurulu tarafından onaylanmış protokol ile, bir hemşire olarak, eğitimli bir tıbbi profesyonel gibi olmalıdır. <ol start="2"><li> , EDTA içeren bi...

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	<li>Chesla, S. E., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+two-dimensional+receptor-ligand+binding+kinetics+by+micropipette.">Measuring two-dimensional receptor-ligand binding kinetics by micropipette.</a> Biophys. J. 75, 1553-1572 (1998).</li><li>Chesla, S. E., Li, P., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+anchor+influences+ligand+binding+two-dimensional+kinetic+rates+and+three-dimensional+affinity+of+FcgammaRIII+(CD16).">The membrane anchor influences ligand binding two-dimensional kinetic rates and three-dimensional affinity of FcgammaRIII (CD16).</a> J. Biol. Chem. 275, 10235-10246 (2000).</li><li>Williams, T. E., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+and+independent+binding+of+Fcgamma+receptors+IIa+and+IIIb+to+surface-bound+IgG.">Concurrent and independent binding of Fcgamma receptors IIa and IIIb to surface-bound IgG.</a> Biophys. J. 79, 1867-1875 (2000).</li><li>Williams, T. E., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+binding+to+multiple+ligands:+kinetic+rates+of+CD16b+for+membrane-bound+IgG1+and+IgG2.">Concurrent binding to multiple ligands: kinetic rates of CD16b for membrane-bound IgG1 and IgG2.</a> Biophys. J. 79, 1858-1866 (2000).</li><li>Williams, T. 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Med. 208, 81-90 (2011).</li><li>Jiang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-stage+cooperative+T+cell+receptor-peptide+major+histocompatibility+complex-CD8+trimolecular+interactions+amplify+antigen+discrimination.">Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.</a> Immunity. 34, 13-23 (2011).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophys. J. 87, 4237-4245 (2004).</li><li>Zhu, C., Williams, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+concurrent+binding+of+multiple+molecular+species+in+cell+adhesion.">Modeling concurrent binding of multiple molecular species in cell adhesion.</a> Biophys. J. 79, 1850-1857 (2000).</li><li>Downey, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+leukocytes+in+capillaries:+role+of+cell+size+and+deformability.">Retention of leukocytes in capillaries: role of cell size and deformability.</a> J. Appl. Physiol. 69, 1767-1778 (1990).</li><li>Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+and+kinetic+analysis+of+Fcgamma+receptor+IIIa+(CD16a)+binding+to+IgG+ligands.">Affinity and kinetic analysis of Fcgamma receptor IIIa (CD16a) binding to IgG ligands.</a> J. Biol. Chem. 282, 6210-6221 (2007).</li><li>Chen, W., Zarnitsyna, V. I., Sarangapani, K. K., Huang, J., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Receptor-Ligand+Binding+Kinetics+on+Cell+Surfaces:+From+Adhesion+Frequency+to+Thermal+Fluctuation+Methods.">Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods.</a> Cell. Mol. Bioeng. 1, 276-288 (2008).</li><li>Thoumine, O., Kocian, P., Kottelat, A., Meister, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+binding+of+fibroblasts+to+fibronectin:+optical+tweezers+experiments+and+probabilistic+analysis.">Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis.</a> Eur. Biophys. J. 29, 398-408 (2000).</li><li>Ounkomol, C., Xie, H., Dayton, P. A., Heinrich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+horizontal+force+probe+for+mechanical+tests+on+pipette-held+cells,+particles,+and+membrane+capsules.">Versatile horizontal force probe for mechanical tests on pipette-held cells, particles, and membrane capsules.</a> Biophys. J. 96, 1218-1231 (2009).</li><li>Piper, J. W., Swerlick, R. A., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+force+dependence+of+two-dimensional+receptor-ligand+binding+affinity+by+centrifugation.">Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.</a> Biophys. J. 74, 492-513 (1998).</li><li>Li, P., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+competition+binding+between+soluble+and+membrane-bound+ligands+for+cell+surface+receptors.">Analysis of competition binding between soluble and membrane-bound ligands for cell surface receptors.</a> Biophys. 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Biri, bazı kritik adımları düşünmelisiniz mikropipet yapışma frekans testinin başarılı bir şekilde kullanın. Birincisi, faiz reseptör-ligand sistemi için spesifik etkileşim kayıt emin olun. Nonspesifik kontrol ölçümleri (bkz. Şekil 3, 4) özgüllük sağlar. İdeal olarak, nonspesifik yapışma olasılıkları tüm temas süresi süreler için 0.05 altında olmalı ve her zaman noktası için spesifik ve nonspesifik yapışma olasılıklar arasında anlamlı bir fark var. Çift eritrosit yüzey liga...

<table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog # </th><th> Yorumlar </th></tr><tr><td> 10x PBS </td><td> BioWhittaker </td><td> 17-517Q </td><td> Kullanmadan önce 1x deiyonize su ile seyreltilir </td></tr><tr><td> Vacutainer EDTA </td><td> BD </td><td> 366643 </td><td> Eritrosit izolasyonu </td></tr><tr><td> 10ML PK100 </td><td></td><td></td><td></td></tr><tr><td> Histopaque 1077 </td><td> Sigma-Aldrich </td><td> 10771 </td><td> Eritrosit izolasyonu </td></tr><tr><td> Adenin </td><td> Sigma-Aldrich </td><td> A2786 </td><td> EAS-45 hazırlık </td></tr><tr><td> D-glikoz (dekstroz) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> EAS-45 hazırlık </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> 6360 </td><td> EAS-45 hazırlık </td></tr><tr><td> Sodyum Klorür (NaCl) </td><td> Sigma-Aldrich </td><td> S7653 </td><td> EAS-45 hazırlık </td></tr><tr><td> Sodyum Fosfat, Dibazik (Na &amp; # x2082; HPO ₄) </td><td> Fisher Scientific </td><td> S374 </td><td> EAS-45 hazırlık </td></tr><tr><td> L-glutamin </td><td> Sigma-Aldrich </td><td> G5763 </td><td> EAS-45 hazırlık </td></tr><tr><td> Biotin-X-NHS </td><td> Calbiochem </td><td> 203188 </td><td> Eritrositlerde biyotinilasyon </td></tr><tr><td> Dimetilformamid (DMF) </td><td> Thermo Scientific </td><td> 20673 </td><td> Eritrositlerde biyotinilasyon </td></tr><tr><td> Borat Tampon (0.1M) </td><td> Elektron Mikroskopi Bilimleri </td><td> 11455-90 </td><td> Eritrositlerde biyotinilasyon </td></tr><tr><td> Streptavidin </td><td> Thermo Scientific </td><td> 21125 </td><td> Functionalizing Ligand </td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A0336 </td><td> Functionalizing Ligand </td></tr><tr><td> Quantibrite PE boncuklar </td><td> BD Biosciences </td><td> 340495 </td><td> Yoğunluk kantifikasyon </td></tr><tr><td> Akış sitometresi </td><td> BD Immunocytometry Sistemleri </td><td> BD LSR II. </td><td> Yoğunluk quanldirimi </td></tr><tr><td> Kılcal Tüp 0.7-1.0mm x 30 &quot; </td><td> Kimble Cam A.Ş. </td><td> 46485-1 </td><td> Mikropipet çekerek </td></tr><tr><td> Madeni Yağ </td><td> Fisher Scientific </td><td> BP2629-1 </td><td> Odası montaj </td></tr><tr><td> Mikroskop Kapak Camlar </td><td> Fisher Scientific </td><td> 12-544-G </td><td> Odası montaj </td></tr><tr><td> PE α-insan CD11a Klon HI 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> Şekil 1 için kimyasalı </td></tr><tr><td> PE anti-insan CD54 </td><td> eBioscience </td><td> 12-0549 </td><td> Şekil 1 için kimyasalı </td></tr><tr><td> Fare IgG1 İzotip Kontrol PE </td><td> eBioscience </td><td> 12-4714 </td><td> Şekil 1 için kimyasalı </td></tr><tr><td> hidrolik mikromanipülatör </td><td> Narishige </td><td> MO-303 </td><td> Mikropipet sistemi </td></tr><tr><td> Mekanik manipülatör </td><td> Newport </td><td> 461-xyz-m, SM-13, DM-13 </td><td> Mikropipet sistemi </td></tr><tr><td> piezoelektrik tercümecisi </td><td> Physik Instrumente </td><td> P-840 </td><td> Mikropipet sistemi </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> Sürüm 8.6 </td><td> Mikropipet sistemi </td></tr><tr><td> DAQ kurulu </td><td> National Instruments </td><td> USB-6008 </td><td> Mikropipet sistemi </td></tr><tr><td> Optik tablo </td><td> Kinetik Sistemleri </td><td> 5200 Serisi </td><td> Mikropipet sistemi </td></tr></tbody></table>

adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

Mikropipet yapışma testinin iki boyutlu (2D) reseptör-ligand bağlayıcı kinetiği 1 ölçmek için 1998 yılında geliştirilmiştir. Tahlil yapışma sensörü ve etkileşen moleküllerin birini sunan hücre gibi bir insan kırmızı kan hücreleri (RBC) kullanır. RBC, tam kontrol ve bağ oluşumunu sağlamak için diğer etkileşim molekülü ifade başka bir hücre ile temas getirmek için mikromanipülasyon kullanmaktadır. Yapışma olayı birbirinden ayrı iki hücre çekerek üzerine RBC uzama olarak algılanır. RBC yüzeyinde hareketsiz ligandlar yoğunluğu kontrol ederek, yapışma olasılığı 0 ile 1 arasında orta menzilli tutulur. Yapışma olasılığı belirli bir temas süresi için iki hücre arasındaki tekrarlanan temas döngülerinin bir dizi frekans adezyon olaylar tahmin edilmektedir. Zamanla değişen bağlayıcı bir eğri oluşturur. Bağlayıcı reseptör-ligand reaksiyon kinetiği 1 olasılıklı model uydurmaeğrisi 2B yakınlık ve kapatma hızı ile döner. Assay, IgG Fc 1-6, selektinler glycoconjugate ligandlar 6-9 ligandlar 10-13, homotypical kaderin bağlama 14, peptid ana histokompatibilite kompleksleri 15 ile T hücre reseptör ve coreceptor integrinler Fcγ reseptörleri etkileşim ile onaylanmıştır 19. Yöntemi, microtopology 5 membran, membran çapa 2, moleküler oryantasyon ve uzunluğu 6, taşıyıcı sertlik 9, eğrilik 20 ve sıkışma kuvveti 20, yanı sıra biyokimyasal faktörler gibi biyofiziksel faktörler, 2D kinetiği düzenlemeleri ölçmek için kullanılan etkileşen moleküllerin bulunduğu iskeleti ve membran mikroçevresinin ve bu moleküller 15,17,19 yüzey örgütü, modülatörler gibi. Bu yöntem t da kullanılır olmuşturo, değiştirilmiş bir modelini 21 kullanarak çift reseptör-ligand türleri 3,4 aynı anda bağlama, ve 19 trimolecular etkileşimleri çalışma . Yöntemin en büyük avantajı, kendi ana membran ortamda reseptörlerinin çalışma izin vermesidir. Saflaştırılmış reseptörleri 17 kullanılarak elde edilen sonuçlar çok farklı olabilir. Ayrıca reseptör-ligand etkileşimleri de tipik biyokimyasal yöntemlerin ötesinde zamansal çözünürlüğe sahip bir alt-ikinci ölçeğinde çalışma sağlar. Mikropipet yapışma frekans yöntemi göstermek için, α L β 2 etkinleştirmek için çözüm dimerik E-selektin ile nötrofil bağlayıcı eritrositlerde integrin α L β 2 Fonksiyonlu hücrelerarası adezyon molekülü 1 (ICAM-1) kinetik ölçüm göstermektedir.

Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

Hem molekülleri etkileşen hücrelerin yüzeylerinde demirlemiş olan reseptör-ligand etkileşimi kinetiği ölçmek için bir yapışma frekans tahlil açıklanmıştır. Bu mekanik tabanlı test mikropipet basınçlı bir insan adezyon sensörü olarak kırmızı kan hücresi ve integrin αLβ2 ve hücrelerarası adezyon molekülü-1 reseptörleri ve ligandlar etkileşim olarak kullanarak örneklenir.

Yapışma için Frekans Testi Yerinde</emHücre-Hücre Arayüz Çapraz Junctional Moleküler Etkileşimler> Kinetik Analizi

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

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Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Yüksek çözünürlüklü Fiber-optik Mikroendoskopi In situ Hücresel Görüntüleme

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Quantitative FRET FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Kantitatif SENP1 Proteaz Kinetik Belirlenmesi (Förster rezonans enerji transferi) Analizi FRET

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Plasenta karşısında Ksenobiyotik ve Nanomalzemelerin Ulaştırma Hızı belirlenmesi kullanarak<em&gt; Ex vivo</em&gt; İnsan Plasental Perfüzyon Modeli

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Için Amelogenin-kitosan hidrojelin geliştirilmesi<em&gt; In Vitro</emBir Yoğun Arayüzü ile&gt; Emaye büyütme

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

TCR-pMHC Bağlama Ölçüm<em&gt; In Situ</em&gt; Bir FRET tabanlı Mikroskopi Assay kullanılarak

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

Tripeptit stabilize Nanoemulsion oleik asit

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

Sinirsel implantları yumuşatma davranışını tahmin etmek için çevre dinamik mekanik analiz

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

İnsan Nötrofil Swarming in vitro Kemotaktik ve Moleküler Analizi için Biyopartikül Mikrodizileri

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

Avustralya Senkrotron'da Seri Kristalografi Ölçümleri için Lipidico Enjeksiyon Protokolü

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

Biyomembran Kuvvet Probu Spektroskopisi ile Moleküler Yay Sabit Analizi

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...

Yapışma için Frekans Testi Yerinde Kinetik Analizi