This protocol allowed the continuous detection of transient effect in the form of some morphological changes during a viral infection, such as detachment and floating of adherent cells. This protocol allowed for a non-invasive and non-labor intensive approach for continuous monitoring cellular morphological changes and capturing transient effect like vascular leakage that could be missed by endpoint assay. This protocol can be used to analyze vascular integrity changes of other cell lines in different experimental setups if the cells are adherent cells.
After preparing the HUVECs, launch the RTCA software application by double clicking the icon on the desktop. Type in the username and password into the login window. Place a 96 well RTCA resistor plate with the A1 well facing inward into the RTCA station located in the cell culture incubator.
Then go to the experiment notes tab of the RTCA software and type the experiment name, device serial number, and device type. In the layout tab under the cell tab of the RTCA software, highlight all the wells, and right click on the highlighted wells to ensure all the wells are turned on. Next, open the schedule tab of the RTCA software.
Click Step_1, and set sweeps to 10 and interval to 30 seconds. Then click start continue in the top left corner of the software to begin the verification run. After the run, open the cell index tab to check the data.
All the cell index values should be less than 0.063. Then check the raw scan data in the lower part of the cell index tab for repeated value patterns as described in the manuscript. To finish the verification run, click plate, and select release plate.
Remove the 96 well RTCA resistor plate from the RTCA station. To obtain background reading on RTCA, wash the collagen type one coated specialized gold microelectrode plate twice with 60 microliters per well of HBSS. Discard the remaining HBSS, and add 50 milliliters per well of extracellular matrix.
Then place the plate into the RTCA station located in the cell culture incubator with the A1 well facing inward. In the exp notes tab of the RTCA software, type in the experiment name, device serial number, and device type. Navigate to the cell tab of the RTCA software and select the layout tab.
Highlight the wells to be used, and right click on the highlighted wells to make sure all the wells are turned on. Now go to the schedule tab of the RTCA software. Click Step_1, and click start or continue in the top left corner to obtain a background reading.
The plate is now ready for cell seeding and removal from the RTCA station. Afterward, seed 10 to the fourth cells per well of HUVECs in the gold microelectrode plate with 50 microliters per well of conditioned medium. Place the plate back into the RTCA station located in the cell culture incubator, with the A1 well facing inward.
Then in the schedule tab of the software, click add a step in the top left corner. Click on Step_2. Change the sweeps to 601, the interval to two minutes, and click apply.
Click on Step_2, and click start or continue in the top left corner of the software. After 20 hours of incubation when the cell index values in the well graph tab show a plateau, the plate is ready for infection. In the schedule tab of the RTCA software, click abort step.
The plate is now ready to be removed from the RTCA station. To infect the HUVECs, retrieve the Zika virus stock from the minus 80 degrees Celsius freezer. Dilute the Zika virus stock with serum-free extracellular matrix in 1.5 milliliter centrifuge tubes to achieve multiplicities of infection of 0.1 and one.
Next, discard the conditioned medium from each well, and rinse each well with 60 microliters of HBSS while using the side of the well. Add 40 microliters of diluted virus sample into the well, starting from the highest to lowest dilution, using the side of the well. Incubate the plate in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide to allow virus absorption.
Swirl the plate five times every 15 minutes to allow virus absorption throughout the cell monolayer. After one hour of incubation, remove and discard the virus suspension from the lowest concentration to the highest. Wash the infected cells twice with 60 milliliters of HBSS.
Overlay the wells with extracellular matrix supplemented with 2%FBS. For the positive control wells, dilute the thrombin in 20 units per milliliter with extracellular matrix supplemented with 2%FBS, and overlay the wells with thrombin containing extracellular matrix medium, using the side of the well. Now click on the schedule tab, and add a step in the top left corner.
Navigate to Step_3, and modify the sweeps to 3, 601, and the interval to two minutes. Click apply, then click OK in the popped up window. Place the infected specialized gold microelectrode plate with the A1 well facing inward into the RTCA station located in the culture incubator.
Finally, click on Step_3, and click start continue in the top left corner of the software. The cell index value of HUVECs infected with Zika virus P6-740 at a multiplicity of infection of 0.1 started to drop at 15 hours post infection, compared to the negative infection control. The HUVECs infected with a higher dose of Zika virus P6-740 at a multiplicity of infection of one showed a drop in the cell index at 11 hours post infection.
At 24 hours post infection, there was a 5%and 7%drop in the normalized cell index value when the endothelial cells were infected at a multiplicity of infection of 0.1 and one respectively. A 50%reduction in the normalized cell index value was recorded at 96 hours post infection and 66.75 hours post infection upon infection at a multiplicity of infection of 0.1 and one respectively. The normalized cell index value reached its lowest point at 107 hours post infection, with a reduction of 51%and 60%upon infection at a multiplicity of infection of 0.1 and one respectively.
The normalized cell index value was found to have a 4%and 13%increase from 107 hours post infection onward until the end of the experiment at 168 hours post infection. Before each RTCA experiment, a resistor verification run using the 96 well RTCA plate is essential to ensure each biosensor works well to produce reliable cell index values.
