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

The work was supported by National Natural Science Foundation of China (U1703118), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Shuangchuang Program, Open Funds of the State Key Laboratory for Chemo/Biosensing and Chemometrics (2016015) and the National Laboratory of Biomacromolecules (2017kf05) and Jiangsu Specially-Appointed Professor project, China.

NOTE: In general, the regeneration process includes trypsin digestion and rinsing step with ethanol and water. The digestion time changes according to the number of cells used, and the type and number of cells used may differ depending on the experimental purposes. It is advised to check the regenerated microchips using optical and electrochemical methods to optimize the regeneration conditions. During the experiment, soluble and insoluble chemicals may be involved, and here these two typical cases of the regeneration pr...

<ol>
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We summarized several available methods17,23,24,25,26 for regenerating microchips in Table 1. Basically, these methods involved relatively harsh experimental conditions to achieve the complete regeneration of chips because of the presence of strong molecule-molecule interactions such as that of the immune complex used for SPR chips. However, t...

Owing to its efficient and less labor-intensive experimental process, label-free cell-based technology has witnessed rapid growth over the past decade for analytical as well as screening purposes such as in the aspect of proteomics1,2, drug delivery3, etc.4,5 Compared with traditional biochemical methods aimed at cell analysis, label-free real-time cell assay with the prototype developed by Giaever and coworkers previously6 is based on the principle of recording electric signal changes on the surface of cell-attached microchips, which permit a continuous measurement of cell growth or migration in a quantitative manner. Following this strategy, a real-time cell electronic sensing (RT-CES) system using electric impedance-based detection principle was introduced7,8 and more recently a commercial real-time cell analyzer (RTCA) was launched for laboratory research9.
The commercial real-time cell analyzer mainly reads out the cells&#39; evoked signals of electric impedance, which result from the physiological changes of incubated cells including cell proliferation, migration, viability, morphology, and adherence on the surface of microchips10,11. Such electric signals are further converted by the analyzer into a dimensionless parameter named the Cell Index (CI) to assess the cell status. The change of the impedance of microchips mainly reflects the local ionic environment of covered cells at the electrode/solution interface. Therefore, the analytical performance of the cell analyzer relies heavily on the core sensing unit, the disposable microchips (i.e., so-called electronic plates, e.g., 96/16/8-well), which are made of arrayed gold microelectrodes lithographically printed at the bottom of incubation wells. The gold microelectrodes assemble in a circle-on-line format (Figure 1) and cover most of the surface area of the incubation wells, which allow for dynamic and sensitive detection of attached cells3,12,13,14. The CI will increase in the case of more surface coverage of cells on the chip, and decrease when cells are exposed to a toxicant resulting in apoptosis. Although the real-time cell analyzer has been frequently used to determine cytotoxicity11 and neurotoxicity15 and provide more kinetic information than classical endpoints method, the disposable electronic plates are the costliest consumable.
Until now, there have been no available methods for the regeneration of the electronic plate, which is probably due to the fact that harsh regeneration conditions, such as piranha solution or acetic acid are involved16,17,18, which may alter the electric status of gold microchips. Therefore, a mild and efficient method to remove the adherent cells and other substances from the surface of gold chips will be desirable for the electronic plate regeneration process. We have recently developed a protocol aimed at the regeneration of disposable electronic plates using non-corrosive reagents and the regenerated chips were characterized by electrochemical as well as optical methods19. By using readily available and moderate laboratory reagents including trypsin and ethanol, we have established a general method to regenerate the commercial electronic plate without adverse effects, which is successfully applied to regenerate the two main types of electronic plate (both 16 and L8) used for RTCA.

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			<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

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 to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

Surface properties of gold microchips: The regeneration procedures used in this protocol were outlined in Figure 1. Figure 2 shows the microscopic surface pictures of fresh and regenerated electronic plates by the optical microscope. As shown in Figure 2A and Figure 2C, microscopic observations indicated that there was virtually no diffe...

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

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

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

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

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