Overview
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.
Protocol
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
- Prepare MDCK (Madin-Darby Canine Kidney) cells in 75 cm2 flasks to obtain freshly split cells (approximately 80% confluence) 24 h before the experiment.
- Wash cells with 5 mL of 1x PBS and detach them by adding 3 mL of 0.25% Trypsin-EDTA solution.
- Add 7 mL of fresh cell culture medium and count cells using an automated cell counter with trypan blue staining.
- 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.
- Leave the E-plate (Table of Materials) at RT for several minutes and add 100 µL of cell culture media to each well using a multi-channel pipette. Do not touch the electrodes of the E-Plate.
- 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.
- Open the software.
- In "Default experiment pattern setup", choose the selected cradle(s) and double-click on the top page, then enter the name of the experiment. Click "Layout" and enter the necessary sample information for each selected well of the plate; then, click "Apply" when finished. Click "Schedule" | "Steps" | "Add a step". The software automatically adds a step of 1 s to measure the background impedance (CI).
- Click on "Start/Continue" in the "Execute" tab. Click on "Plot", add all samples by selecting the appropriated wells, and ensure CI is between -0.1 and 0.1 before proceeding to the next step.
- Remove the plate from the cradle.
- Add 100 µL of each cell suspension from step 1.4 in duplicate to the appropriate wells and 100 µL 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.
- Insert the E-plate into the cradle pocket. Click "Schedule" | "Add step" in the software and enter values to monitor cells every 30 min for 200 repetitions. Then, select "Start/Continue".
- Check and plot the CI data by clicking on the "Plot" 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.
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Materials
Name | Company | Catalog Number | Comments |
E-Plate 16 (6 plates) | ACEA Biosciences, Inc | 5469830001 | E-plates are available in different packaging |
PBS 1X | Life technologies (gibco) | 14040091 | |
TPCK-Trypsin | Worthington | LS003740 | |
xCELLigence Real-Time Cell Analysis Instrument S16 | ACEA Biosciences, Inc | 380601310 | The xCELLigence RTCA S16 instruments are available in different formats (16-well, 96-well, single or multi-plate) |
75 cm2 tissue culture flask | Falcon | 430641U | |
MEM 1X | Life technologies (gibco) | 31095029 |