Biz Dr teşekkür ederim. John Hancock, Ray Grill, Andrew Morris ve verdikleri destek için Olga Chumakova. FlexStation 3 sisteminin UTHSC Cytodynamic Görüntüleme Tesis bir parçası. Ayrıca Dr teşekkür. Scripps Araştırma Enstitüsü ve HEK293-hTRPA1 istikrarlı hücre hattı sağlamak için Novartis Araştırma Vakfı (GNF) Genomik Enstitüsü Ardem Patapoutian ve Michael Bandell. Araştırma NIH (MXZ için GM081658) ve Texas Tıp Merkezi Sindirim Hastalıkları Merkezi (HH) ve Mission Bağlan / TIRR Vakfı (HH) hibe kısmen desteklenmektedir.

1. 96-kuyucuğu Hücre hazırlıkları CDNA insan TRPA1 geçici trasfected HEK293 hücreleri için <ol><li> HEK293 hücrelerin% 10 ısı ile inaktive edilmiş fetal sığır serumu (Invitrogen, 16.000-044) ile desteklenmiş 4.5 mg / ml glikoz (Thermo Scientific, SH3002201) içeren Dulbecco&#39;nun Minimal Essential Orta 1-2 gün (DMEM), 50 adet için yetiştirilen / ml penisilin ve 50 mg / ml streptomisin (Invitrogen, 15.140-163). Transfeksiyon gününde, hücre yoğunluğu% 70-90 ulaştırılması gerekmektedir. </li><li> Iyi 50 ul poli-L-ornitin (Sigma P3655, steril distile su içinde 20 mg / ml) ve 37 kuluçka ° C, en az 15 dakika boyunca pipetleme Coat 96-kuyucuğu. Dulbecco&#39;nun Fosfat her biri de bir kez durulayın Mg 2 olmadan Tamponlu Salin (DPBS) + ve Ca 2 + (Thermo Scientific, SH3002803). Plaka, birkaç saat için hücre kültürü kaputu sol olabilir. </li><li> 96 plaka her kuyu için, 1.5 ml Eppendorf tüp 25 ul OPTI-MEM (Invitrogen, 31.985-070) 0.2 mg cDNA seyreltin. Aynı cDNA transfekte kuyu sayısına göre orantılı olarak büyütmek. Ayrıca 25 ul OPTI-MEM transfekte olmak için her sıra ayrı bir 1.5 ml Eppendorf tüp 0.4 ul Lipofectamine 2000 (Invitrogen, 11.668-019) sulandırmak. Transfekte için kuyu toplam sayısına göre bu kadar Ölçeği. </li><li> Kısaca vorteks seyreltilmiş cDNA ve Lipofectamine 2000 ve bunları yaklaşık 5 dakika oda sıcaklığında bekletin. </li><li> Eşit hacimde sulandırılmış Lipofectamine 2000 seyreltilmiş cDNA, girdap içeren tüp transferi ve karışımı 15 dakika ila 2 saat oda sıcaklığında bekletin. Bu süre zarfında, 49 ul / poli-L-ornitin kaplı plaka Lipofectamine / cDNA 2000 karışımı aktarın. </li><li> HEK293 hücreleri (Adım 1.1) Trypsinize, bir hemasitometre veya Kontes otomatik hücre sayıcı (Invitrogen, C10227) kullanarak hücre yoğunluğu sayımı, ve 15 ml steril bir hücre istenilen miktarda transfer (genellikle yaklaşık ~ 125,000-150,000 hücre / kuyu) alt konik santrifüj tüpüne. 5 dakika için 1000 x RPM santrifüjleyin. % 10 ısı ile inaktive edilmiş fetal sığır serumu ile takviye DMEM kültür ortamı ~ 1,250,00-1,500,000 hücre / ml, ancak antibiyotik olmadan yeniden süspanse hücre. </li><li> Bir çok kanallı pipetter veya MicroFill Mikroplaka Dispenseri (Bio-Tek) kullanarak 2000 cDNA-Lipofectamine karışımı içeren her bir kuyunun 98 ul hücre süspansiyonu koyun. Plaka nemlendirilmiş hücre kültürü inkübatör koymadan önce en az 30 dakika kaputu oturup edelim. Için 37 ° C,% 5 CO 2 ile 20-44 saat inkübe edin . </li></ol> Stably TRPA1 ifade HEK293 hücreleri için <ol start="8"><li> Adım 1.2 kat 96-kuyucuğu izleyin. </li><li> Cömertçe indüklenebilir HEK293-hTRPA1 hücre hattı (Invitrogen, Dr. A. Patapoutian (Scripps Araştırma Enstitüsü) tarafından sağlanan ve yabani tip HEK293 hücreleri (Adım 1.1) olarak aynı ortamda muhafaza ancak 5 mg / ml blasticidin ile desteklenmiş A1113902) ve 50 mg / ml hygromycin B (Invitrogen, 10.687.010). Hücre yoğunluğu yaklaşık% 90 ulaştığında, hücreler trypsinize hücre yoğunluğu saymak ve 5 dakika boyunca 1000 x RPM hücreler istenilen miktarda santrifüj. ~ 1.000.000 hücre / ml yoğunlukta doksisiklin (Fisher, BP26535, 0.25 mg / ml) ile takviye aynı kültür ortamında süspanse edin hücreleri. </li><li> Ul / iyi, çok kanallı pipetter veya MicroFill (Bio-Tek) kullanarak 100 poli-L-ornitin kaplı plakalar hücre süspansiyonu koyun. Plaka nemlendirilmiş hücre kültürü inkübatör koymadan önce en az 30 dakika kaputu oturup edelim. 37 ° ° C&#39;de,% 5 CO 2 - 16 24 saat. </li></ol> 2. Çözümler <ol><li> Hank tamponlu tuz solüsyonu (HBSS), ekstrasellüler çözüm olarak kullanılır. (MM) içerir: 137 NaCl, 5.4 KCl, 0.25 Na 2 HPO 4, 0.44 KH 2 PO 4, 1.3 CaCl 2, 1.0 MgSO 4, 1.0 pH 1N NaOH ile 7.4 &#39;e ayarlanır MgCl 2, 10 glikoz, 10 Hepes,). </li><li> Fluo-4 tahlil tampon HBSS içinde probenesid 2 mM içerir. 0,5 N NaOH 250 mM stok probenesid (Sigma, P8761) olun. HBSS için 1:125 bir oranında seyreltilir. 1 N HCl ile pH 7.4 yeniden ayarlayınız. </li></ol> 3. Hücre yükleme Fluo-4 AM <ol><li> 1 mM stok solüsyonu 912 ul susuz DMSO 1 mg Fluo 04:00 (Invitrogen, F14202) eritin. Fluo-4 en az bir ay için stok çözelti -20 ° C&#39;de saklanabilir AM. </li><li> 10 ul 1 mM karıştırın Fluo-4 10 ul Pluronic sonra F-127 (Invitrogen, P3000MP, DMSO içinde% 20 (w / v) solüsyonu) ve 5 ml Fluo-4 tahlil tamponu (Adım 2.2) tüm içeriği transfer ile AM (Sigma, A9418),% 0,1 Bovine serum albümin ile desteklenmiştir. </li><li> Mikroplak yıkayıcı (Bio-Tek gelen ELx405TM gibi) kullanarak 80 ul / 2 kez HBSS (Adım 1.7 veya 1.10) 96-kuyucuğu yetiştirilen hücreler yıkayın. </li><li> Fluo-4 Ul / çok kanallı bir pipetter veya MicroFill (Bio-Tek) kullanarak ve 37 plaka inkübe 50 (Adım 3.2) çözümü yükleme PM ° C 1 saat. </li><li> ELx405TM mikroplak yıkayıcı kullanarak Fluo-4 tahlil tampon (Adım 2.2.) Hücreleri 3 kez yıkayın. Son yıkamadan sonra, her bir kuyunun Fluo-4 tahlil tampon 80 ul bırakın. </li></ol> 4. Bileşik plaka hazırlanması <ol><li> 1 saat hücre yükleme dönemde, 3x Fluo-4 tahlil tampon istenilen son konsantrasyonlarının agonistleri (veya antagonistleri) 3x dilüsyonları bir dizi hazırlamak. </li><li> 96-iyi bir bileşik plaka seyreltilmiş bileşik çözümler Pipet 250-300 ul. FlexStation 3, bir defada tek bir sütun okur çünkü mümkün olduğunda bileşik plaka aynı sütun ve ilgili pozitif ve negatif kontroller bir bileşik seri dilüsyonları ettirmelisiniz. </li></ol> 5. Plaka okuma FlexStation 3 <ol><li> (FlexStation 3, bilgisayar ve SoftMax Pro 5.2 yazılım) sistemi açın </li><li> Deney yeni bir dosya adı ile kaydedin. Set-up, ESNEK modunu seçin ve aşağıdaki değerleri girin: </li></ol> Protokol # 1, agonist etkisi <img src="/files/ftp_upload/3149/3149table1.jpg" alt="Tablo 1" /> Protokol # 2, antagonist etkisi <img src="/files/ftp_upload/3149/3149table2.jpg" alt="Tablo 2" /><ol start="3"><li> Yeni bir uç kutusu, bileşik plakası ve plaka okuma, 3 FlexStation uygun tepsilere yerleştirin ve &quot;oku&quot; butonuna tıklayın. FlexStation 3 Bir kerede bir sütun okumak ve önceden programlanmış zaman noktalarında bileşikler okuma kesmeden katacak. Bir sütun ve makine adım 5.2 &#39;de kullanıcı tarafından programlanan tüm sütunlar okunur kadar yapılan bileşik eklemelerle sonraki sütun okumak için devam edecektir okuma bitirmek için yaklaşık 3 dakika (Protokol # 2 için 5 dakika) sürer. </li></ol> 6. Veri analizi <ol><li> Deneysel veriler SoftMax Pro 5.2 yazılımını kullanarak doğrudan veya kopyalanan ve Microsoft Excel gibi herhangi bir elektronik tablo programı içine yapıştırılan işlenebilir. TRPA1 agonist flufenamic asidi (FFA) konsantrasyon-yanıt eğrisi aşağıdaki en küçük kareler uydurma rutin kullanarak inşa edilmiştir. <img src="/files/ftp_upload/3149/3149eq1.jpg" alt="Denklem 1" /> Y gözlenen tepki olduğu, Y max normalize edilmiş maksimum tepki, C, ilgili ilaç konsantrasyonu, EC 50, yarı-maksimum bir tepki uyarılmış konsantrasyon ve n H görünürdeki Hill katsayısı. Varsayılan TRPA1 antagonisti bileşik Konsantrasyon-inhibisyon eğrisi FFA (100 mcM) sabit bir konsantrasyonu ve kademeli olarak aşağıdaki uyan en küçük kareler ile hesaplanan bileşik bileşik IC 50 değeri A. konsantrasyonu arttırarak elde edilir denklemi. <img src="/files/ftp_upload/3149/3149eq2.jpg" alt="Denklem 2" /> , Burada gözlenen yanıt Y ve Y maksimum pik yanıt normalize [Ant] karşılık gelen bileşik A toplama, IC 50 FFA-uyarılmış yanıt yarı-maksimum inhibisyonu üreten antagonist konsantrasyonu ve n H görünürdeki Tepesi katsayısı. </li></ol> 7. Temsilcisi Sonuçlar: TRPA1 + duyarlı boya Ca 2, Fluo-4 (1-2) ile tespit edilebilir kalsiyum akınına yol açar aktivasyonu Ca 2 +-geçirgen katyon kanal,. HEK293-hTRPA1 istikrarlı bir hücre hattı TRPA1 agonistleri ve antagonistleri konsantrasyon-yanıt ilişkileri incelemek için kullanılmıştır. Hücreler, Fluo-04:00 yüklenen 80 ul tahlil tampon yerleştirilir ve FlexStation 3 kullanılarak okunabilir. 30 s için temel floresan kaydettikten sonra, bir seri seyreltme TRPA1 agonist, FFA 40 ul, her istenilen nihai konsantrasyonu 3x akışkansal modülünün bir parçası olarak dahil bir multi-kanal pipetter aracılığıyla hücrelere ilave edildi FlexStation 3. Floresan değişiklikler ek bir 180 s. monitörize edildi Sırasıyla F ve F 0 faiz ve okuma başında zaman floresan değerlerdir - (F 0 F), her bir kuyu için göreceli Fluo-4 floresan değişiklikleri mesafe kadar taşınmış = ifade edildi. FFA konsantrasyonlarının bir dizi yanıt floresan değişikliklere zamanlı kurslar Temsilcisi izleri Şekil. 1. Maksimal yanıt düzeyleri benzer olmasına rağmen, alt FFA konsantrasyonlarda daha yüksek aktivasyon kinetiği açıkça daha hızlıdır, son dört FFA konsantrasyonları için dikkat edin. Bu nedenle, bu farklılıkları ayırt etmek için, konsantrasyon-yanıt ilişkisi üzerine ilk floresan artış oranları (eğim) FFA değişik konsantrasyonlarda (Şekil 2) ek ölçüm tarafından inşa edilmiştir. Yarım maksimal etkisiFFA ive konsantrasyonu (EC 50) 55.4 mcM (n = 3) olarak tespit edilmiştir. Varsayılan TRPA1 antagonisti test etmek için, bir bileşik, protokol # 2 (Adım 2) kullanarak benzer bir deney gerçekleştirdi. Bu durumda, bir dizi antagonisti dilüsyonlarının 30 s (3x istenilen son konsantrasyonlarının 40 ul) ilave edildi ve FFA (100 mcM son bir konsantrasyon için 300 mcM 60 ul) sonra 180 ler eklendi. Bileşik bir doz-yanıt bağımlı 4.3 mcM yarım maksimal inhibitör konsantrasyonu (IC 50) (n = 3) (Şekil 2) ile FFA TRPA1 hücreleri azalır. <img src="/files/ftp_upload/3149/3149fig1.jpg" alt="Şekil 1"/> FFA insan TRPA1 Şekil 1. Konsantrasyon bağımlı aktivasyonu. İzler zamanla floresan FFA-uyarılmış artış (sadece ilk 60 s tepkiler FFA-uyarılmış yanıtlar hızlı kinetiği daha iyi göstermek için gösterilmiştir). Yüksek konsantrasyonlarda FFA ile floresan artış uyarılmış olduğunu lütfen unutmayınız çok düşük konsantrasyonlarda daha hızlı kinetik, zirve floresan seviyeleri benzer olmasına rağmen. <img src="/files/ftp_upload/3149/3149fig2.jpg" alt="Şekil 2"/> Şekil 2 Konsantrasyon - FFA indüklenen floresan değişim ve bileşik stably HEK293 hücrelerinde ifade insan TRPA1 FFA yanıtı indüklediği inhibisyonu için tepki ilişkileri. Δ, konsantrasyon - FFA yanıt ilişkisi üzerine ilk normalize floresan artış oranları (eğim) FFA konsantrasyonları değişen ek ölçerek tespit edildi. O, varsayılan TRPA1 antagonisti FFA-uyarılmış kalsiyum yanıt inhibisyonu Konsantrasyon-yanıt ilişkisi, bileşik A.

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Çıkar çatışması ilan etti.

Hücre içi Ca 2 +, nörotransmitter sürüm (3), kardiyak aksiyon potansiyeli (4), kas hücresi kasılma (5), hücre sinyal iletimi (6-7) ve eksitotoksik hücre ölümü (gibi pek çok fizyolojik ve patolojik koşullar, önemli bir rol oynar 8). Hücre içi Ca 2 + seviyeleri ölçümü fonksiyonel değişiklikler ve Ca 2 + geçirgen kanallar plazma zarı ya da hücre içi organelleri katkısı aydınlatmak için yardımcı olur Ca 2 + yukarıdaki süreçleri (9-13). Ta...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> FlexStation 3 </td><td> Moleküler Cihazlar </td><td> FLEX3 </td><td></td></tr><tr><td> 96-Peki, FlexStation pipetleyin İpuçları </td><td> Moleküler Cihazlar </td><td> 9000-0912 </td><td></td></tr><tr><td> 96-Peki Tabaklar </td><td> Greiner Bio-one </td><td> 655090 </td><td></td></tr><tr><td> 96-Peki Temizle Düz Taban plakaları </td><td> Costar </td><td> 3595 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12430-104 </td><td></td></tr><tr><td> Opti-MEM </td><td> Invitrogen </td><td> 31985-070 </td><td></td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-019 </td><td></td></tr><tr><td> PM fluo-4 </td><td> Invitrogen </td><td> F14202 </td><td></td></tr><tr><td> PluronicF-127 </td><td> Invitrogen </td><td> P3000MP </td><td></td></tr><tr><td> Poli-L-ornitin </td><td> Sigma </td><td> P36551 </td><td></td></tr><tr><td> probenesid </td><td> Sigma </td><td> P8761 </td><td></td></tr><tr><td> Sığır serum albumini </td><td> Sigma </td><td> A9418 </td><td></td></tr></tbody></table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

Moleküler Aygıtlar FlexStation 3, çok iyi plakaları otomatik floresan ölçüm yeteneğine bir masa üstü multi-mode mikroplaka okuyucu. Akademik ayarları orta-yüksek verimli ekranlar için idealdir. Bu bir multi-kanal pipetter ve makinenin bir defada bir çok floresan reaktiflerin floresan değişiklikleri izlemek için bir sütun okur ile donatılmış entegre bir sıvı aktarım modülü vardır. Örneğin, FlexStation 3 Ca 2 + geçirgen iyon kanalları ve G-protein reseptörler hücre içi serbest Ca 2 + düzeyleri değişiklikleri ölçerek işlevini incelemek için kullanılmıştır. Geçici reseptör potansiyeli (TRP) kanalları, pek çok fizyolojik ve patofizyolojik fonksiyonlarında önemli rol oynamaktadır seçici olmayan katyon kanalları büyük bir aileyiz. TRP kanalları çoğu kalsiyum geçirgen ve etkinleştirme sırasında kalsiyum akını neden. Bu video, TRPA1 kanal, çok sayıda Ağrılı uyaranlara için bir moleküler sensör farmakolojik profil çalışma FlexStation 3 uygulama göstermektedir. Geçici ya da stabil bir şekilde 96-kuyucuğu yetişen insan TRPA1 kanal, ifade HEK293 hücreleri floresan boya + duyarlı Ca 2, Fluo-4, ve bu hücrelerin gerçek zamanlı floresan değişiklikler ile yüklenen uygulama öncesi ve sırasında ölçülen FlexStation 3 FLEX modunu kullanarak bir TRPA1 agonist. Varsayılan TRPA1 antagonisti etkisi de incelenmiştir. Veri SoftMax Pro yazılımı TRPA1 aktivatörleri ve inhibitörleri konsantrasyon-yanıt ilişkileri oluşturmak için transfer edilir.

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Bu video FlexStation 3 kullanarak insan TRPA1 kanalları farmakolojik profili çalışmak için ayrıntılı bir protokol sağlar. Protokol hücre hazırlama, boya yükleme ve mikroplaka okuyucu, FlexStation 3 operasyon detayları kapsar.

FlexStation kullanarak 3 TRP Kanal İşlevleri Yüksek Verimli Tarama Orta Hücre tabanlı Kalsiyum Testi

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

cell-based-calcium-assay-for-medium-to-high-throughput-screening-trp

Research

JoVE Journal

Bioengineering

21.0K Görüntüleme.  The University of Texas Health Science Center at Houston. Bu video FlexStation 3 kullanarak insan TRPA1 kanalları farmakolojik profili çalışmak için ayrıntılı bir protokol sağlar. Protokol hücre hazırlama, boya yükleme ve mikroplaka okuyucu, FlexStation 3 operasyon detayları kapsar.

Video: Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Biyomalzemelerin Yüksek Verimli Discovery için Polimer Mikroarray&#39;ler

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Biyomühendislik

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Yüksek verim Antifungal İlaç Tarama Candida albicans Biyofilm Chip (Ca BChip)

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Bir mikroakışkan Platform kullanarak Yüksek throughput Protein Ekspresyonu Jeneratör

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

Biyolojik Sıvı Ortamda Ölçeklenebilir, Metalik Yüksek Aspect Ratio Nanokompositlerin Üretimi

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Tomoauto kullanma: Bir Protokol Yüksek verim Otomatik Cryo-elektron Tomografi için

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Tıbbi Cihazlar Bakterilerin yüksek verimlilik Kimlik Savar Polimerler

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Enjekte edilebilir rejeneratif terapi ve yüksek-den geçerek uyuşturucu tarama için 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Çin Hamster Yumurtalık Hücre Yetiştiriciliğinde Yüksek İş-İş BulaMetli Otomatik Mikro-Biyoreaktörler Kullanılarak Proses Optimizasyonu

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Pseudomonas syringae pv Direnci yüksek-Throughput Tanımlama. Fide Sel Asası Kullanarak Domates

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Yüksek Verimli Mikroakışkan Cihaz Kullanarak Dinamik ÇevreSel Koşulların Üretilmesi

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...