Encyclopedia of Experiments: Biology
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Transcript
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 "Default experiment pattern setup," highlight the selected cradles and double-click on the top page to enter the name of the experiment. Click "Layout" and enter the necessary sample information for each selected well of the plate. Click "Schedule" | "Steps" |, and "Add a Step." The software will automatically add a 1-second step to measure the background impedance.
Click "Execute" and "Start / Continue." Click "Plot" and "Add" 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 "Schedule" and "Add Step," and enter values to monitor the cells every 30 minutes for 200 repetitions before selecting "Start / Continue."
To check and plot the cell impedance data, click "Plot" 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.
