Methods for various particle quantification represent a critical aspect of most biology studies. This protocol allows a precise quantification of the viral data in real time. This technique replaces the traditional measurement of viral data.
By objective realtime data, we are below our confidence interval, avoiding labor-intensive endpoints assessed. This method can be used for all viruses that induce a cytopathic effect. Demonstrating the procedure will be Quentin Grassin, a laboratory technician from our laboratory.
To propagate and amplify H1N1 viruses, seed 7.5 times 10 to the 6 MDCK cells on two 75-square-centimeter tissue culture flasks according to standard protocols, and incubate the cells for 24 hours at 37 degrees Celsius to reach 90 to 100%confluence. The next day, wash the cells two times with five milliliters of sterile PBS per flask per wash, and label one flask as the control. Next, dilute a thawed vial of influenza A virus stock to the appropriate experimental concentration in a 1.5-milliliter tube containing fresh virus propagation medium.
Use one milliliter of the diluted stock to infect the MDCK cells at a multiplicity of infection of 1 times 10 to the negative 3, or 1 times 10 to the negative 4 plaque-forming units per cell, and add one milliliter of virus propagation medium to the control flask. Adsorb the virus to the cells for 45 minutes at room temperature, with stirring every 15 minutes. At the end of the incubation, remove the inoculum and replace it with 15 milliliters of virus propagation medium supplemented with one microgram per milliliter of TPCK trypsin to facilitate cleavage of viral hemagglutinin HA0 into HA1 and HA2 subunits.
Then place the flask in the 35-degree-Celsius incubator for three days. At the end of the incubation, compare the cells in each culture under a 40x objective on a light microscope to assess the cytopathic effects on the cells. When the cytopathic effect is complete, add the cell culture supernatants to a 15-millimeter tube and collect the cellular debris from each culture by centrifugation.
Then, transfer the clarified virus culture supernatant to a 15-milliliter tube, and aliquot the progeny viruses to single-use sterile cryogenic vials for minus 80 degrees Celsius storage. 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 three milliliters of 0.25%Trypsin-EDTA per flask at 37 degrees Celsius. When the cells have detached, stop the reaction with seven milliliters of fresh culture medium per flask, and count the cells from each culture.
Dilute the cells to 4 times 10 to the 5th cells per milliliter of cell culture medium and perform twofold 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 one-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 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. To determine the correlation between the CIT50 values and the multiplicity of infection, after culturing 3 times 10 to the 4th freshly-split MDCK cells into each well of the electronic microtiter plate for 24 hours, wash the cells two times with 100 microliters of fresh virus propagation medium per well, per wash, and use a single-channel pipette to add 100 microliters of viral suspension to each well. When all of the virus dilutions have been added, gently load the plate into the cradle pocket of the instrument at 35 degree Celsius, and begin monitoring the cell impedance every 15 minutes for at least 100 hours, as demonstrated.
After two cycles of measurements, click to pause the apparatus, and remove the E-plate from the cradle. Add 100 microliters of virus propagation medium supplemented with TPCK trypsin to each well and return the E-plate into the cradle pocket. Then, start the analysis.
To assess the influenza A virus survival kinetics, add sodium chloride to a final concentration of 35 grams per liter in distilled water, and add 900 microliters of the resulting saline solution to two-milliliter cryo-tubes. Add 100 microliters viral stock to each cryo-tube and place the tubes in the 35-degree-Celsius incubator for the appropriate experimental time period. The day before the end of the incubation, seed 3 times 10 to the 4th freshly split MDCK cells and 100 microliters of virus propagation medium per well in a 16-well microtiter plate in repeat steps, allowing measurement of the background impedance and uniform distribution of the cells onto the bottom of the wells.
Then grow the cells for 24 hours at 37 degree Celsius and 5%carbon dioxide. The next day, wash the cells twice, then infect the cells with 100 microliters of the saline-distilled virus diluted 10 times in fresh culture medium, as demonstrated, and monitor the cell impedance every 15 minutes for at least 100 hours. Here, raw data obtained after 120 hours with different concentrations of MDCK cells are shown.
After 24 hours, cell index measurements revealed that cells in wells seeded with 3 times 10 to the 4th cells were still in the exponential phase of growth, therefore, this cell concentration was used for subsequent experiments. MDCK cell cultures demonstrate a clear linear relationship between the CIT50 and the initial multiplicity of infection by influenza virus. The results from a typical survival kinetic experiment show a decrease in the cell index due to virus-induced cytopathic effect.
The CIT50 can be used to calculate the viral inactivation slope for each virus in each condition for the determination of which virus had the greatest stability in the studied environment. The stability of the virus indirectly correlates to the inactivation slope, allowing, for example, amino acid residues in the hemagglutinin glycoprotein that are involved in influenza A virus survival outside the host to be identified. This technique is very sensitive to small variations.
Therefore, using an automatic cell counter is encouraged in order to obtain reproducible results between experiments. This method can also be used to compare the replication of different viruses, to investigate the virus tropism for several cell lines at a time, or to study specific steps of the virus cycle.
