Cellular Impedance-Based Survival Kinetics Assay to Assess Virus Survival

1. Preparation of reagents and starting materials

1. Preparation of MDCK cells and sterile cell culture medium
   1. Cultivate Madin-Darby Canine Kidney (MDCK) cells in modified Eagle's medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin).
   2. After thawing, passage MDCK cells at least 2x before infecting them to ensure complete recovery (but less than 30 passages to avoid any drift in cell phenotypes).
   3. Seed 75 cm²  tissue culture flask(s) containing 30 mL of sterile 1x MEM with 7.5 x 10⁶  MDCK cells and incubate at 37 °C in humidified 5% CO₂ incubator.
2. Preparation of impedance monitoring equipment
   1. Place the instrument in the incubator at 35 °C and allow it to warm up for at least 2 h.
   2. Connect it to the control unit placed outside the incubator.
   3. Proceed to the cleaning procedures as recommended by the manufacturer before starting the remainder of the experiment.
3. Production of IAV stocks on MDCK cells
   1. To propagate and amplify H1N1 viruses, seed 7.5 x 10⁶ MDCK cells on two 75 cm² tissue culture flasks, and incubate for 24 h at 37 °C to reach 90%–100% confluence.
   2. Decant the cell culture medium from the cell monolayer in 75 cm² flasks. Wash the cells with 5 mL of sterile 1x PBS.
   3. Remove PBS and add 5 mL of 1x PBS to wash the cells again.
   4. Label one flask as the control and remove PBS before adding 15 mL of virus propagation media (1x MEM with 0% FCS) carefully on the monolayer. Incubate this flask in an incubator maintained at 35 °C with 5% CO₂. Use it for comparison after 3 days of propagation.
   5. Thaw one vial of IAV stock at RT. Dilute the virus to the appropriate concentration in a 1.5 mL tube containing virus propagation media (1x MEM with 0% FCS).
   6. Remove 1x PBS from the 75 cm² flask and infect MDCK cells at a multiplicity of infection (MOI) of 1 x 10^-3 or 1 x 10^-4 plaque-forming units per cell (pfu/ cell) by adding 1 mL of the diluted virus to the cell monolayer.
   7. Adsorb virus to MDCK cells for 45 min at RT by stirring the flask regularly every 15 min.
   8. Gently remove the inoculum and add 15 mL of virus propagation media per flask containing 1 µg/mL TPCK-trypsin (trypsin/L-1-tosylamide-2- phenylethyl chloromethyl ketone), to cleave the viral hemagglutinin HA0 into HA1 and HA2 subunits (an event that is required for HA fusion with the endosomal membrane and release of the viral genome).
   9. Incubate the flasks at 35 °C and 5% CO₂ for at least 3 days to replicate the virus.
   10. Observe MDCK cells under a microscope at 40x magnification and look for cytopathic effects (CPE) on the cells (by comparing them to the cell control flask). If CPE is not complete (i.e., around 80% of the cells are detached from the substrate), put the flasks back in the incubator for an additional 24 h.
   11. When CPE is complete, decant the cell culture supernatant and centrifuge at 300 x g for 10 min to pellet the cellular debris.
   12. Transfer the clarified supernatant to a 15 mL tube and aliquot progeny viruses to single-use sterile cryogenic vials. Immediately place the cryotubes at -80 °C to freeze and stock viruses.
4. Infect MDCK cells with different 10-fold dilutions of a known 6 log₁₀ TCID₅₀/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 in step 2.10.
   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 1 µg/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.

2. IAV survival kinetics

1. Expose IAV to saline distilled water at 35 °C and test for their infectivity over time by measuring cell impedance decrease.
   1. Prepare saline distilled water by adding NaCl to a final concentration of 35 g/L in distilled water. Add 900 µL of saline water into 2 mL cryotubes.
   2. Add 100 µL of viral stock in saline water and place the cryotubes in an incubator (35 °C, 5% CO₂) for 1 h, 24 h, or 48 h.
   3. Seed 100 µL (containing 3 x 10⁴) of freshly split MDCK cells on a 16-well microtiter plate and grow for 24 h at 37 °C and 5% CO₂.
   4. Infect cells with 100 µL of exposed viruses (previously diluted 10x in culture media) using the reverse pipetting method for reproducibility by repeating section 1.4.
2. Monitor cell impedance every 15 min for at least 100 h.