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

Arbeidet ble støttet av National Natural Science Foundation of China (U1703118), et prosjekt finansiert av prioritet faglig Program utvikling av Jiangsu høyere utdanning institusjoner (PAPD), Jiangsu Shuangchuang programmet, åpne midler av nøkkelen tilstand Laboratorium for kjemoterapi/Biosensing og kjemometri (2016015) og National Laboratory av Biomacromolecules (2017kf05) og Jiangsu Specially-Appointed Professor prosjektet, Kina.

Merk: vanligvis gjenfødelse prosessen inkluderer trypsin fordøyelsen og skyllingsprosess trinn med etanol og vann. Fordøyelsen tiden endres av antall celler som brukes, og type og antall celler brukes kan variere avhengig av de eksperimentelle formålene. Det anbefales å sjekke de gjenfødte microchips bruker optisk og elektrokjemiske metoder for å optimalisere gjenfødelse betingelsene. Under eksperimentet løselig og uløselig kjemikalier kan være involvert, og her disse to typiske tilfeller av gjenfødelse prose...

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Vi oppsummert flere tilgjengelige metodene17,23,24,25,26 for regenererende microchips i tabell 1. Innerst inne, disse metodene involvert relativt hardt eksperimentelle forhold å oppnå hele fornyelse av chips på grunn av sterk molekyl-molekylet interaksjoner som immun komplekset brukes for SPR sjetonger. Disse harde eksperimentelle forhold k...

På grunn av sin effektive og mindre arbeidskrevende eksperimentelle prosessen, etikett-fri cellen-basert teknologi har vært vitne til rask vekst det siste tiåret for analytisk samt sortering formål som i aspektet av Proteomikk1,2 , stoffet levering3, etc.4,5 forhold med tradisjonelle biokjemiske metoder rettet mot celle analyse, etikett-gratis sanntid celle analysen med prototypen utviklet av Giaever og kolleger tidligere6 er basert på prinsippet om innspillingen elektrisk signal endringer på overflaten av celle-vedlagt microchips, som tillater en kontinuerlig måling av cellevekst eller migrering i en kvantitativ måte. Etter denne strategien, en sanntids celle elektronisk sensing (RT-CES) systemet bruker elektrisk impedans-basert oppdagelsen prinsippet var introdusert7,8 , og mer nylig en kommersiell sanntid celle analyzer (RTCA) ble lansert for laboratorium forskning9.
De kommersielle sanntid celle analyserer hovedsakelig leser ut cellene vakte signaler av elektrisk impedans, som følge av fysiologiske endringer inkubert celleområde inkludert celle spredning, migrasjon, levedyktighet, morfologi og etterlevelse på den overflaten av microchips10,11. Slike elektriske signaler konverteres ytterligere av analyseringen til en dimensjonsløs parameter kalt celle indeks (CI) å vurdere celle status. Endring av impedans på microchips gjenspeiler hovedsakelig ioniske lokalmiljøet dekket celler på elektroden/løsning grensesnittet. Derfor stoler analytisk ytelsen av cellen analysator tungt på kjernen sensing enheten, de disponible microchips (dvs., såkalte elektronisk plater, f.eks, 96/16/8-vel), som er laget av plassert gull microelectrodes lithographically skrives ut nederst på inkubasjon brønner. De gull microelectrodes sammen i en sirkel-on-line format (figur 1) og dekker det meste av arealet av inkubering, som tillater dynamisk og følsom deteksjon av tilknyttede celler3,12,13 ,14. CI vil øke hvis flere dekning av celler på chip, og redusere når celler er utsatt for et toxicant som resulterer i apoptose. Selv om sanntid celle analysatoren er ofte brukt til å bestemme cytotoksisitet11 og nevrotoksisitet15 og gi kinetic mer enn klassisk endepunktene metoden, er disponibel elektronisk platene det costliest Forbruksvare.
Til nå, har det ikke vært noen metoder for fornyelse av den elektroniske platen, som er trolig på grunn av at harde gjenfødelse forhold, som piranha løsning eller eddiksyre er involvert16,17, 18, som kan endre elektrisk status for gull microchips. En mild og effektiv metode for å fjerne tilhenger cellene og andre stoffer fra overflaten av gull chips vil derfor være ønskelig for elektronisk plate gjenfødelse prosessen. Vi har nylig utviklet en protokoll rettet mot fornyelse av disponibel elektronisk plater ved hjelp av ikke-korroderende reagenser og gjenskapte chips var preget av elektrokjemiske samt optiske metoder19. Ved hjelp av lett tilgjengelige og moderat laboratorium reagenser inkludert trypsin og etanol, har vi etablert en generell metode for å regenerere den kommersielle elektroniske platen uten bivirkninger, som er brukt til å regenerere de to hovedtypene elektronisk plate (16 og L8) brukes for RTCA.

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			<td style="width:317px;">Invitrogen</td>
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			<td height="21" style="height:21px;width:293px;">Alexa Fluor Donkey anti-goat 488</td>
			<td style="width:317px;">Invitrogen</td>
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		</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

Etikett-fri cellebasert analysen er fordelaktig for biokjemiske studien fordi det ikke krever bruk av forsøksdyr. På grunn av sin evne til å gi mer dynamisk informasjon om cellene under fysiologiske forhold enn klassisk biokjemiske analyser, tiltrekker denne etiketten-gratis sanntid celle analysen basert på elektrisk impedans mer oppmerksomhet i løpet av siste tiår. Dens praktisk utnyttelse kan imidlertid være begrenset på grunn av relativt dyre kostnadene for måling, der dyre forbruksvarer disponible gull microchips brukes for den celle analysatoren. I denne protokollen, har vi utviklet en generelle strategi for å regenerere plassert gull microelectrodes utstyrt for en kommersiell etikett-fri celle analysator. Trypsin fordøyelsen, skylling med etanol og vann og en spinnende trinn omfatter prosessen med gjenfødelse. Den foreslåtte metoden er testet og vist seg å være effektive for regenerasjon og gjentatt bruk av kommersielle elektronisk plater minst tre ganger, som vil hjelpe forskere lagre på den høye kjørende kostnaden på sanntid celle analyser.

Overflateegenskaper av gull microchips: gjenfødelse prosedyrer brukes i denne protokollen ble skissert i figur 1. Figur 2 viser den mikroskopiske overflaten bilder av friske og regenerert elektronisk plater av optisk mikroskopet. Som vist i figur 2A og figur 2C, mikroskopiske observasjoner indikerte at det var nesten ingen forskjell i...

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

Denne protokollen beskriver en generell strategi for å regenerere kommersielle plassert gull microelectrodes utstyrt for en etikett-fri celle analysator å spare på høy løpende kostnader ofmicrochip-baserte analyser. Gjenfødelse prosessen inkluderer trypsin fordøyelsen, skylling med etanol og vann og en spinnende trinn, som gjør at gjentatt bruk av microchips.

Regenerering av plassert gull Microelectrodes utstyrt for en sanntid celle Analyzer

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

Svært Sensitive og Rapid Fluorescence Detection med en bærbar FRET Analyzer

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

En Protein Forberedelse metode for High-throughput identifisering av proteiner samspill med et kjernefysisk kofaktor Bruke LC-MS / MS-analyse

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

Sporing av DNA-fragmenter i sanntid etter Smartphone

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

Et modifisert gjær-ett hybrid system for Heteromer Proteinkompleks-DNA-interaksjonsstudier

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

En fluorescens svingninger spektroskopi analysen av Protein-Protein interaksjoner i celle-celle kontakter

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

Sanntid observasjon av DNA Strand Exchange reaksjonen formidlet av Rad51

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

Real-time, semi-automatisert fluorescerende måling av luftveiene overflaten flytende pH av primær menneskelige Luftveisepitel celler

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

Overvåking GPCR-β-arrestin1/2 interaksjoner i Real time levende systemer for å akselerere Drug Discovery

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

Bruke en ekstracellulære Flux Analyzer til å måle endringer i Glykolysen og oksidativt fosforylering under Mouse sperm Capacitation

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

Måling av nukleotidbinding til intakte, funksjonelle membranproteiner i sanntid

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