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

Protocollo

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

1. Prepare MDCK (Madin-Darby Canine Kidney) cells in 75 cm² flasks to obtain freshly split cells (approximately 80% confluence) 24 h before the experiment.
2. Wash cells with 5 mL of 1x PBS and detach them by adding 3 mL of 0.25% Trypsin-EDTA solution.
3. Add 7 mL of fresh cell culture medium and count cells using an automated cell counter with trypan blue staining.
4. 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.
5. 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.
6. 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.
7. Open the software.
   1. 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).
   2. 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.
8. Remove the plate from the cradle.
9. 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.
10. 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".
11. 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.

Materiali

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