Impedance-Based Cellular Assay to Measure Multiplicity of Infection of Influenza Virus

1. Correlation between CIT₅₀ values and multiplicity of infection

1. Add 100 µL of sterile 1x MEM culture medium in each well of the E-plate. Insert the E-plate into the cradle pocket of the instrument at 35°C.
2. Measure the background as described:
3. 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 1s 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.
4. Remove the E-plate from the cradle.
5. Seed 3 x 10⁴ of freshly split MDCK cells on each well of the electronic microtiter plate and grow them for 24h at 35°C with 5% CO₂ so that they are in a replicative phase during IAV infection.
6. Infect MDCK cells with different 10-fold dilutions of a known 6log₁₀TCID₅₀/mL titer of H1N1 virus, using reverse pipetting for reproducibility by following the steps below:
   1. Rinse MDCK cells 2x with 100 µL of MEM with no FCS (virus propagation media). Be aware of removing all media after the second wash to avoid further dilution of the inoculum.
   2. Add 100 µL of viral suspension in each well using a single-channel pipette. To avoid contamination, proceed by starting from left to right, then top-to-bottom in the plate while covering the remaining wells with the lid.
   3. Insert the plate into the cradle pocket of the instrument at 35°C. Be gentle to avoid sudden movements potentially leading to contamination.
   4. Start to monitor cell impedance every 15 min during at least 100 h as described:
      1. 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".
   5. After the two cycles of measurements (i.e., 30 min), pause the apparatus by clicking on "Pause" in the "Execute" tab and remove the E-plate from the cradle.
   6. Add 1µg/mL TPCK-trypsin to the virus propagation media to cleave the viral hemagglutinin.
   7. Add 100 µL of virus propagation media containing TPCK-trypsin into each well and insert the E-plate into the cradle pocket.
   8. Click "Start/Continue" in the "Execute" tab.
      NOTE: Do not forget to create a negative control corresponding to mock-infected cells by replacing viral suspension with virus propagation media.