cellular impedance-based analysis for monitoring of cellular processes in real-time

Cellular Impedance-Based Analysis for Monitoring of Cellular Processes in Real-Time

To determine the appropriate cell quantity for cell infection, prepare 24-hour, approximately 80% confluent MDCK cell cultures, as demonstrated, before washing the cells with PBS, and harvesting them with a 45-minute incubation in 3 milliliters of 0.25% trypsin-EDTA per flask at 37 degrees Celsius.
When the cells have detached, stop the reaction with 7 milliliters of fresh culture medium per flask, and count the cells from each culture. Dilute the cells to 4 x 105 cells per milliliter of cell culture medium, and perform two-fold serial dilutions of the cells, as indicated.
Place an E-plate at room temperature for several minutes, before adding 100 microliters of cell culture medium to each well without touching the electrodes of the E-plate. Unlock the cradles and insert the plate front end into the cradle pocket of the impedance-measuring instrument. Close the door of the incubator, and open the software.
In the &#34;Default experiment pattern setup,&#34; highlight the selected cradles and double-click on the top page to enter the name of the experiment. Click &#34;Layout&#34; and enter the necessary sample information for each selected well of the plate. Click &#34;Schedule&#34; | &#34;Steps&#34; |, and &#34;Add a Step.&#34; The software will automatically add a 1-second step to measure the background impedance.
Click &#34;Execute&#34; and &#34;Start / Continue.&#34; Click &#34;Plot&#34; and &#34;Add&#34; to select the appropriate wells, confirming that the background impedance is between negative 0.1 and 0.1, before proceeding to the next step.
Next, remove the plate from the cradle and add 100 microliters of each cell suspension to the appropriate wells in duplicate. Leave the E-plate in the laminar flow hood for 30 minutes at room temperature to allow a uniform distribution of the cells onto the bottoms of the wells, before loading the E-plate into the cradle pocket. Click &#34;Schedule&#34; and &#34;Add Step,&#34; and enter values to monitor the cells every 30 minutes for 200 repetitions before selecting &#34;Start / Continue.&#34;
To check and plot the cell impedance data, click &#34;Plot&#34; and select the concentration of cells that is just before the stationary phase 24 hours after seeding, to obtain cells that are still in a growing phase during the viral infection.

cellular-impedance-based-analysis-for-monitoring-cellular-processes

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

EoE Sub Articles

eoe sub articles

NOTE: During all experiments, keep the plates on non-electrostatic surfaces at all times, such as the paper wraps from the packaging. Follow section 1 below to determine the most appropriate concentration of cells to be seeded in the electronic microtiter plate (designed as E-Plate).
1. Determination of appropriate cell quantity for infecting cells
<ol>
	<li>Prepare MDCK (Madin-Darby Canine Kidney) cells in 75 cm2 flasks to obtain freshly split cells (approximately 80% confluence) 24 h before the experiment.</li>
	<li>Wash cells with 5 mL of 1x PBS and detach them by adding 3 mL of 0.25% Trypsin-EDTA solution.</li>
	<li>Add 7 mL of fresh cell culture medium and count cells using an automated cell counter with trypan blue staining.</li>
	<li>Adjust the cell concentration to 400,000 cells/mL with cell culture media. Perform two-fold serial dilutions in additional tubes to obtain cell densities of 200,000; 100,000; 50,000; 25,000; 12,500; and 6,250 cells/mL. Adjust the dilution range of the cells according to the cell type and their growth behavior.</li>
	<li>Leave the E-plate (Table of Materials) at RT for several minutes and add 100 &#181;L of cell culture media to each well using a multi-channel pipette. Do not touch the electrodes of the E-Plate.</li>
	<li>Unlock the cradles and insert the plate front end into the cradle pocket of the impedance measuring instrument (Table of Materials). Close the door of the incubator.</li>
	<li>Open the software.
	<ol>
		<li>In &#34;Default experiment pattern setup&#34;, choose the selected cradle(s) and double-click on the top page, then enter the name of the experiment. Click &#34;Layout&#34; and enter the necessary sample information for each selected well of the plate; then, click &#34;Apply&#34; when finished. Click &#34;Schedule&#34; | &#34;Steps&#34; | &#34;Add a step&#34;. The software automatically adds a step of 1 s to measure the background impedance (CI).</li>
		<li>Click on &#34;Start/Continue&#34; in the &#34;Execute&#34; tab. Click on &#34;Plot&#34;, add all samples by selecting the appropriated wells, and ensure CI is between -0.1 and 0.1 before proceeding to the next step.</li>
	</ol>
	</li>
	<li>Remove the plate from the cradle.</li>
	<li>Add 100 &#181;L of each cell suspension from step 1.4 in duplicate to the appropriate wells and 100 &#181;L of cell media in wells used as controls. Leave the E-plate in the laminar flow hood for 30 min at RT to allow for uniform distribution of the cells at the bottoms of the wells.</li>
	<li>Insert the E-plate into the cradle pocket. Click &#34;Schedule&#34; | &#34;Add step&#34; in the software and enter values to monitor cells every 30 min for 200 repetitions. Then, select &#34;Start/Continue&#34;.</li>
	<li>Check and plot the CI data by clicking on the &#34;Plot&#34; button in the software. Select the concentration of cells that are just before the stationary phase 24 h after seeding on the plate, in order to obtain cells that are still in a growing phase during viral infection. Stationary phase is reached when CI is at its maximum.</li>
</ol>

<table><tbody><tr><td>E-Plate 16 (6 plates)</td><td>ACEA Biosciences, Inc</td><td>5469830001</td><td>E-plates are available in different packaging</td></tr><tr><td>PBS 1X</td><td>Life technologies (gibco)</td><td>14040091</td><td></td></tr><tr><td>TPCK-Trypsin</td><td>Worthington</td><td>LS003740</td><td></td></tr><tr><td>xCELLigence Real-Time Cell Analysis Instrument S16</td><td>ACEA Biosciences, Inc</td><td>380601310</td><td>The xCELLigence RTCA S16 instruments are available in different formats (16-well, 96-well, single or multi-plate)</td></tr><tr><td>&amp;nbsp;75 cm2 tissue culture flask</td><td>Falcon</td><td>430641U</td><td></td></tr><tr><td>MEM 1X</td><td>Life technologies (gibco)</td><td>31095029</td><td></td></tr></tbody></table>

Source: Labadie, T., et al., <a href="https://app.jove.com/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the process of real-time monitoring of cellular events, including the proliferation of adherent cells, via a cellular impedance assay. Here, the adherent cells act as insulators, restricting the current flow between the electrodes and increasing the impedance.

Source: Labadie, T., et al., Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis. J. Vis. Exp. (2021)
In this video, we ...

For the continuous measurement of cell behavior using real-time cell analysis, RTCA, obtain an RTCA microtiter plate containing impedance measuring-gold microelectrode arrays embedded in the well bottom. Carefully add cell culture medium to the wells.
Within the electrically conductive medium, apply a weak electrical potential across the microelectrodes. The potential causes electric current to flow between the microelectrodes, facilitating the measurement of the baseline impedance &#8212; the medium&#39;s inherent electrical resistance.&#160;
Pipette the desired concentrations of mammalian cell suspension to the medium-containing wells. Allow the cells to uniformly distribute in the well.
During incubation, the cells adhere onto the well bottom.&#160; The adherent cells on the microelectrodes act as an insulator, restricting the current flow and increasing the impedance.
Gradually, the cells proliferate on the microelectrodes, leading to increased exponential impedance.
Following confluent cell layer formation, the cells encounter nutrient deprivation and undergo death. As a result, the cells detach from the microelectrodes, decreasing the impedance.
The continuous impedance measurement facilitates real-time monitoring of the cellular events, including cell proliferation and death.

53 ビュー. 細胞プロセスをリアルタイムでモニタリングするための細胞インピーダンスベースの解析

Cellular Impedance-Based Analysis for Monitoring of Cellular Processes in Real-Time

記録

Cellular Impedance-Based Analysis for Monitoring of Cellular Processes in Real-Time