An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Day 1

NOTE: Perform all the steps described below in a sterile laminar flow biosafety cabinet used for tissue culture in a BSL-2 laboratory.

1. Thaw the serum samples at room temperature and transfer 100 µL of each serum sample to a sterile tube. Heat inactivate for 30 min at 56°C either in a water bath or a temperature-adjustable thermomixer. Make 10-fold serial dilutions of the serum sample ranging from 1:1 to 1:1,000 using cold RPMI-10 (RPMI with 10% fetal bovine serum).
2. Transfer 10 µL of serially diluted serum sample to each well of a sterile 96-well V-bottom plate. Include two sets of control wells, RPMI-10 with RVPs only and without serum sample, and RPMI-10 only without RVPs and without serum sample. Set up each serum sample and RPMI-10 controls in triplicates.
3. Remove the Dengue-1, 2, 3 and 4 RVPs from the -80 °C freezer and thaw them in a 37 °C water bath. Transfer the thawed RVPs immediately to ice. Obtain approximately 170 µL RVPs for each serum sample (10 µL of RVPs x 3 wells for each serum dilution (1:1, 1:10, 1:100, 1:1,000) and 10 µL of RVPs x 3 wells for each of the RPMI-10 with RVP only control wells).
4. Pipette 10 µL of RVPs into each well of the 96-well V-bottom plate containing the serum sample and to the RPMI-10 with RVPs (no serum) control wells. Do not add RVPs to the RPMI-10 only (no serum sample and no RVP) wells. Mix thoroughly by pipetting up and down 5–10 times. Add 10 µL cold RPMI-10 in lieu of RVPs to each cold RPMI-10 only (no serum sample and no RVP) control wells.
5. Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hour at 37 °C in the presence of 5% CO₂. While the 96-well V-bottom plate is incubating, clean the surface of the biosafety cabinet with 70% ethanol and run the UV light for 15 min.
6. Remove a fully confluent T75 flask of K562 cells from the incubator, mix the cells well using a sterile 5 mL pipet, and transfer 5 mL of cells to a sterile 15 mL conical tube.
   NOTE: The K562 cells are maintained in RPMI-10 and subcultured at 1 x 10⁶/mL.
7. Count the number of cells by removing 10 µL cells from the sterile 15 mL conical tube and mix with 10 µL tryphan blue. Count the cells using a hemocytometer to determine the total number of cells in the 15 mL conical tube.
8. Centrifuge the 15 mL conical with cells at ~1,200 x g for 10 minutes, decant the supernatant, and resuspend the cells in warm RPMI-10 at a concentration of 80,000 cells/30 µL of media (2.66 x 10⁶ cells /mL).
9. Remove the 96-well V-bottom plate from the incubator (step 1.6), transfer 30 µL of K562 cells to each well of the 96-well V-bottom plate, and mix thoroughly by pipetting up and down 5–10 times. Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hr at 37 °C in the presence of 5% CO₂.
10. After incubating for 1 h, remove the 96-well V-bottom plate from the incubator and centrifuge at ~1,200 x g for 5 minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10% bleach.
11. Wash the cells in each well by resuspending them in 125 µL of warm RPMI-10, mix thoroughly with a pipette, and centrifuge the plate at ~1,200 x g for 5 min. Decant the media by turning the plate upside down into a container containing 10% bleach. Repeat this wash step two times.
12. After washing, add 100 µL of warm RPMI-10 to each well, mix up and down with the pipet, and incubate the plate for 48 h at 37 °C in the presence of 5% CO₂.

2. Day 3

1. Remove the 96-well V-bottom plate containing 100 µL of cells and media/well from the incubator and move it to a biosafety cabinet. Using a multichannel pipette, mix the contents of each well and transfer the cells and media to a 96-well U-bottom plate or pre-labeled 5 mL polypropylene tubes.
2. Rinse each well in the 96-well V-bottom plate with 100 µL of 1% Paraformaldehyde (PFA) in 1x Phosphate Buffered Saline (PBS) and transfer to the respective wells in the 96-well U-bottom plate or labeled 5 mL polypropylene tubes from step 2.1 to yield a final concentration of 0.5% PFA/well or tube. Mix thoroughly using a multichannel pipette, cover the plate with aluminum foil, and let it sit in the incubator for 30 minutes to fix the cells.
3. Prepare the flow cytometer (see Table of Materials for example) by running unstained K562 cells to calibrate the side and forward scatter along with the fluorescence settings. The only fluorescence channel required is FL1, as GFP is the only fluorescence emitted from the cells. Acquire ~30,000–50,000 cells from each sample.
   NOTE: Any flow cytometer capable of reading a single fluorescence can be used for acquiring the data, as cells infected with RVPs will only emit green fluorescence due to the presence of GFP, and no other fluorescence channels are needed.

3. Data Analysis

1. Analyze data acquired on the flow cytometer using any flow cytometry analysis software (see Table of Materials).
   1. Set the first gate based on FSC-A (forward scatter – area) vs FSC-H (forward scatter – height) to include single cells and exclude auto-fluorescent doublets from the analysis. Then, gate singlet-gated cells based on SSC-A (side scatter – area) vs FSC-A to exclude any autofluorescent debris.
   2. Analyze SSC-A vs FSC-A gated cells for the expression of GFP based on SSC-A vs GFP-A. Determine the percentage of GFP+ cells for each dilution of the serum and control samples by setting a gate around GFP+ cells.
2. Determine the average percentage of GFP+ cells for each serum sample dilution and control wells by dividing the total percentage of GFP+ cells in the triplicate wells by 3.
3. Calculate fold-enhancement of infection by dividing the average percentage of GFP+ cells in the serum samples for each dilution by the average percentage of GFP+ cells in the RPMI-10 with RVPs (no serum sample) control wells.
4. Graph fold-enhancement (y-axis) versus dilution (x-axis), and perform statistical analysis using ANOVA followed by Tukey's post-hoc test for multiple comparisons.