Name: regeneration of arrayed gold microelectrodes equipped for a real-time cell analyzer
Uploaded: 2018-03-12T12:30:00
Description: Watch this Scientific Journal Video about Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer at JoVE.com

İş Ulusal Doğa Bilimleri Vakfı Çin (U1703118), öncelik akademik Program Geliştirme, Jiangsu yüksek öğretim kurumları (PAPD), Jiangsu Shuangchuang Program, devlet anahtarının açık fonlar tarafından finanse edilen bir proje tarafından desteklenmiştir Laboratuar kemoterapi/Biosensing ve Sharky (2016015) ve Biomacromolecules Ulusal Laboratuvarı (2017kf05) ve Jiangsu Specially-Appointed Profesör projesinin, Çin.

Not: genel olarak, tripsin sindirim ve etanol ve su ile durulama adım yenilenme süreci içerir. Sindirim zaman kullanılan hücre sayısına göre değişen ve türüne ve kullanılan hücre sayısına bağlı olarak deneysel amaçlar farklı olabilir. Rejenerasyon koşulları optimize etmek için optik ve elektrokimyasal yöntemleri kullanarak rejenere mikroçip kontrol etmelerini rica ederiz. Deney sırasında çözünür ve çözünmez kimyasallar tutulabilir ve burada bu iki tipik durumda bir yenilenme prosedürü ...

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Biz mikroçip Tablo 1' deki yeniden oluşturulması için çeşitli kullanılabilir yöntemleri17,23,24,25,26 özetlenebilir. Temel olarak, bu yöntemleri cips komple yenilenme güçlü molekül molekül immün kompleks SPR yongaları için kullanılan bu gibi etkileşimleri varlığı nedeniyle elde etmek için nispeten sert deneysel koşulla...

Sayesinde onun verimli ve daha az emek yoğun deneysel işlem, etiket içermeyen hücre tabanlı teknoloji proteomik1,2 açıdan gibi analitik hem de eleme amaçlı son on yılda hızlı büyüme tanıklık etmiş , ilaç teslim3, vb4,5 ile karşılaştırıldığında geleneksel biyokimyasal yöntemlerle hücre analizi, etiket ücretsiz gerçek zamanlı hücre tahlil Giaever ve arkadaşları tarafından daha önce geliştirilen prototip6 ile yönelik elektrik sinyal değişiklikleri nicel bir şekilde sürekli ölçümü hücre büyümesini veya geçiş izni hücre bağlı mikroçip yüzeyinde kayıt prensibi temel alır. Bu stratejiyi, gerçek zamanlı hücre tanıttı7,8 ve son zamanlarda bir ticari gerçek zamanlı hücre analyzer (RTCA) (RT-CES) sistem elektrik empedans tabanlı algılama ilkesini kullanarak oldu algılama elektronik başlatıldı Laboratuvar araştırma9için.
Ticari gerçek zamanlı hücre analyzer ağırlıklı olarak hangi inkübe hücre hücre çoğalması, geçiş, canlılığı, morfoloji ve bağlılık dahil fizyolojik değişiklikler sonucu hücre uyarılmış sinyalleri, elektrik empedans, okur. yüzey mikroçip10,11. Böyle elektrik sinyalleri daha fazla hücre dizin (hücre durumu değerlendirmek için CI) adlı bir boyutsuz parametre Çözümleyicisi tarafından dönüştürülür. Empedans mikroçip değişikliği esas olarak kapalı hücre elektrot/çözüm arabirimi, yerel iyonik ortam yansıtır. Bu nedenle, hücre analyzer analitik performansını dayanır ağır çekirdek algılama birimi üzerinde dizilmiş altın microelectrodes kılan tek kullanımlık kristallerden mikroçipler (Yani, sözde elektronik plakaları, örneğin, 96/16/8-iyi), lithographically kuluçka kuyu dibinde basılmış. Altın microelectrodes daire-on-line biçiminde (şekil 1) birleştirin ve ekli hücreleri3,12,13 dinamik ve hassas tespiti için izin kuluçka Wells yüzey alanının en kapsayacak ,14. CI çip hücrelerinin daha fazla yüzey kapsama alanı söz konusu olduğunda artırın ve hücre içinde apoptosis kaynaklanan bir toxicant maruz azaltın. Her ne kadar gerçek zamanlı hücre analyzer sitotoksisite11 ve nörotoksisite15 belirlemek ve klasik bitiş noktaları yöntemi daha fazla Kinetik bilgi sağlamak için sık sık kullanılmıştır, tek kullanımlık elektronik costliest tabaklar tüketim maddesi.
Şimdiye kadar muhtemelen gerçeğini dahil16,17, piranha çözüm veya Asetik asit gibi sert rejenerasyon koşulların olması nedeniyle olan elektronik plaka rejenerasyon için kullanılabilir hiçbir yöntem olmuştur. Hangi altın mikroçip elektrik durumunu değiştirebilir 18. Bu nedenle, hafif ve etkili bir yöntem yapışık hücreleri ve diğer maddeler altın chips yüzeyinden kaldırmak için elektronik plaka rejenerasyon işlemi için arzu olacak. Biz son zamanlarda aşındırıcı reaktifler kullanarak tek kullanımlık elektronik plakaları yenilenme amaçlı bir protokol geliştirdik ve rejenere cips elektrokimyasal yanı sıra optik yöntemleri19tarafından karakterize. Tripsin ve etanol gibi laboratuar kolayca kullanılabilir ve ılımlı reaktifler kullanarak, biz iki ana türü yeniden oluşturmak için başarıyla uygulanan ticari elektronik plaka olmadan yan etkiler, yeniden oluşturmak için genel bir yöntem kurduk Elektronik plaka (16 ve L8) kullanılan RTCA için.

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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Trypsin&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# T9935</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">DMEM/F12</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 21041-02</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Penicillin/ Streptomycin</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 15140-122</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Glutamine</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 25030-081</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">N-acetyl cysteine&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# A9165</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Insulin</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# 16634</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Apo-transferrin&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# T1147</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Sodium selenite</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# S-5261</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">DMEM (high glucose)</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 11960-044</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Dapi Fluoromount-G</td>
			<td style="width:317px;">Southern Biotech</td>
			<td style="width:263px;">Cat# 0100-20&#160;</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Poly-D-Lysine&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# A-003-E</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Primaria Plates&#160;</td>
			<td style="width:317px;">VWR</td>
			<td style="width:263px;">Cat# 62406-456</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Stock reagents&#160;</td>
			<td style="width:317px;">reconstitution&#160;</td>
			<td style="width:263px;">Concentration used</td>
			<td style="width:192px;">Storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Apo-transferrin</td>
			<td style="width:317px;">10 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">N-acetyl cysteine</td>
			<td style="width:317px;">5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Sodium selenite</td>
			<td style="width:317px;">2.5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:25,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Collagen IV</td>
			<td style="width:317px;">200 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">TGF-b2</td>
			<td style="width:317px;">20 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:10,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">IL-34</td>
			<td style="width:317px;">100 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ovine wool cholesterol</td>
			<td style="width:317px;">1.5 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Heparan sulfate</td>
			<td style="width:317px;">1 mg/mL in H2O</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Oleic acid/Gondoic acid</td>
			<td style="width:317px;">Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparin</td>
			<td style="width:317px;">50 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Solutions&#160;</td>
			<td style="width:317px;">Recipe&#160;</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Comments</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Perfusion Buffer</td>
			<td style="width:317px;">50 &#956;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +)</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Douncing Buffer</td>
			<td style="width:317px;">200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Panning Buffer</td>
			<td style="width:317px;">2 mg/mL of peptone from milk solids in DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Microglia Growth Medium (MGM)</td>
			<td style="width:317px;">DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen IV Coating</td>
			<td style="width:317px;">MGM containing 2 &#956;g/mL collagen IV</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Myelin Seperation Buffer</td>
			<td style="width:317px;">9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Mix well after the addition of&#160; CaCl2 and MgCl2</td>
		</tr>
		<tr height="235">
			<td height="235" style="height:235px;width:293px;">TGF-b2/IL-34/Cholesterol containing growth media (TIC)&#160;</td>
			<td style="width:317px;">MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.&#160;</td>
		</tr>
	</tbody>
</table>

regeneration of arrayed gold microelectrodes equipped for a real-time cell analyzer

Çünkü biyokimyasal çalışma deneysel hayvan kullanımını gerektirmez için etiket içermeyen hücre tabanlı tahlil avantajlıdır. Klasik biyokimyasal deneyleri daha hücreleri fizyolojik koşullar altında hakkında daha dinamik bilgi sağlamak yeteneği nedeniyle, elektrik empedans ilkesine dayanarak bu etiket ücretsiz gerçek zamanlı hücre tahlil geçmişte sırasında daha fazla ilgi topluyor on yıl. Ancak, onun pratik kullanımı nispeten pahalı maliyet pahalı sarf tek kullanımlık altın mikroçip hücre analyzer için kullanılan ölçü nedeniyle sınırlı olabilir. Bu protokol için bir ticari etiket içermeyen hücre analyzer için donatılmış dizilmiş altın microelectrodes yeniden oluşturmak için genel bir strateji geliştirdik. Rejenerasyon işlemi tripsin sindirim, etanol ve su ve bir iplik adım ile durulama içerir. Önerilen yöntem test edilmiş ve en az üç kez, yenilenme ve ticari elektronik plakaların tekrarlanan kullanımı için etkili olduğu gösterilmiştir hangi gerçek zamanlı hücre deneyleri yüksek çalışan maliyeti üzerinde Kaydet araştırmacılar yardımcı olacaktır.

Altın mikroçip yüzey özellikleri: Bu protokol için kullanılan rejenerasyon yordamlar şekil 1' de özetlenen. Şekil 2 mikroskobik yüzey taze resimlerini ve elektronik plakaları optik mikroskop tarafından yeniden gösterir. Şekil 2A ve şekil 2C, mikroskobik gözlemler olduğunu hemen hemen ima etti gösterildiği gibi mikro?...

Watch this Scientific Journal Video about Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer at JoVE.com

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

Bu iletişim kuralı ticari dizilmiş altın microelectrodes ofmicrochip tabanlı yüksek çalışan maliyeti deneyleri tasarruf amaçlı bir hücre etiket ücretsiz analyzer için donatılmış yeniden oluşturmak için genel bir strateji açıklar. Rejenerasyon işlemi tripsin sindirim, etanol ve su ve mikroçip tekrarlanan kullanımı sağlar bir iplik adım ile durulama içerir.

Rejenerasyon için gerçek zamanlı hücre Analyzer donatılmış dizilmiş altın Microelectrodes

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

Research

JoVE Journal

Biochemistry

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Taşınabilir FRET Analyzer ile son derece hassas ve hızlı Floresan Algılama

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

LC-MS / MS Analizi Nükleer kofaktör ile etkileşimde proteinlerin yüksek verimli belirlenmesi için bir protein Hazırlama Yöntemi

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Akıllı Telefon ile DNA Parçalarının Ayırılmasının Gerçek Zamanlı İzlenmesi

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Heteromerik Protein Kompleksi-DNA Etkileşim Çalışmaları İçin Modifiye Maya-Bir Hibrid Sistem

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Bir floresan dalgalanma spektroskopisi tahlil Protein-Protein etkileşimlerinin hücre-hücre kişiler

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

Rad51 tarafından aracılı DNA Strand Exchange tepki gerçek zamanlı gözlem

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

Primer ınsan hava yolu epitel hücrelerinin hava yolu yüzeyi sıvı pH 'Sı gerçek zamanlı, yarı otomatik floresan ölçümü

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Monitoring GPCR-ÃƒÅ½Ã‚Â²-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

İzleme GPCR-β-arrestin1/2 etkileşimleri gerçek zamanlı yaşam sistemleri Ilaç Discovery hızlandırmak için

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Fare Sperm Kapasasyonu Sırasında Glikoliz ve Oksidatif Fosforilasyondaki Değişiklikleri Ölçmek için Hücre Dışı Akı Analizörü Kullanma

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

Sağlam, fonksiyonel membran proteinlerine bağlanan nükleotidin gerçek zamanlı olarak ölçülmesi

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...