An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

1. Preparation of target cells for infection

1. Seed target cells the day prior to performing the experiment in a 96-well plate (the 'target' plate).
2. Plate sufficient cells per well to ensure an almost confluent (~90%) monolayer.
   NOTE: This will depend on the relative size of your target cell type. If using human foreskin fibroblasts (HFFs), seed 20,000 cells/well.
3. Typically, 21 wells (7 down by 3 across) should be seeded per sample, allowing 4 samples to be analyzed per plate.
4. Seed an additional 12 wells per plate (i.e., row 8) to serve as controls.

2. Preparation of virus: antibody mixtures

1. Add 90 µL of media combined with antibody (and well mixed) to create twice the desired initial test antibody concentration into 3 wells of row 1 of a fresh 96 well plate (the 'dilution' plate).
   NOTE: A typical starting concentration of antibody would be 20 µg/mL but should be determined empirically.
2. Add 60 µL of media to each of the 18 wells below (i.e., 6 down by 3 across) the 3 wells from step 2.1.
3. Add 60 µL of media to 12 additional wells (i.e., row 8). 
4. Transfer 30 µL of antibody dilution from row 1 to the corresponding wells in row 2.
5. Mix the contents of row 2 by pipetting smoothly up and down 20 times with the multichannel pipette. 
6. Transfer 30 µL of antibody dilution from row 2 to row 3.
7. Repeat this process of mixing and transferring until the contents of row 7 have been mixed.
8. Once row 7 has been mixed, dispose of 30 µL from the wells in row 7.
9. Dilute HCMV in a complete medium so that 50 µL contains sufficient infectious particles to result in a MOI=1 infection if added directly to the cells.
   NOTE: HCMV is a Biosafety Level 2 (BSL-2) category pathogen. Ensure proper safety procedures are in place and use filter tips for all HCMV-exposed media.
   NOTE: This requires the same number of infectious particles as there are cells/well, which will vary based on cell type and virus tropism. If using HFFs as described above, the required virus concentration is 4×10⁵ pfu/mL.
   NOTE: Ensure a sufficient excess of diluted virus (e.g., 10% extra) is prepared.
10. Add 60 µL of diluted virus stock to all wells containing antibody dilutions, and 6 wells that contain only 60 µL media.
11. Add 60 µL of media only to the remaining 6 wells of media only, to serve as uninfected controls.
12. Incubate the resulting virus: antibody mixtures at 37^oC for 1 h.

3. Infection of target cells

1. Remove all media from target cells.
2. Using a multichannel pipette, transfer 100 µL of virus: antibody mixture from the ‘dilution’ plate onto the cells in the equivalent position on the 'target' plate.
3. Incubate the cells at 37 °C, 5% CO₂ for 24 h.

4. Fixation of cells

1. Remove all media from cells.
   NOTE: There is no need to change tips between wells at this point.
2. Wash all wells with 200 µL phosphate-buffered saline (PBS) and remove.
3. Add 180 µL -20 ^oC, 100% ethanol to all wells.
4. Incubate plate at -20 ^oC for at least 30 min.
   NOTE: This step can be extended to at least one month, provided additional ethanol is added as it begins to evaporate.
   NOTE: The plate is safe to handle under BSL-1 conditions from this point onwards, and non-filter tips can be used. 

5. Detection of virally infected cells by immunofluorescence (IF)

1. Remove ethanol from the plate.
2. Wash all wells with 200 µL PBS and remove.
3. Add 200 µL PBS to all wells and incubate for 5 min.
   1. Meanwhile, prepare primary antibody dilution in PBS.
   2. 100 µL of 1:2000 mouse monoclonal anti-IE will be required per well (note dilution is for the specific antibody listed in materials).
      ​NOTE: Dilution of the antibody used for IF may need optimizing if an alternative is used.
   3. Vortex mixture to ensure sufficient mixing.
4. Remove PBS from all wells and add 100 µL primary antibody dilution.
5. Incubate at room temperature for 1 h.
6. Remove primary antibody from all wells.
7. Add 200 µL PBS and incubate for 5 min.
   1. Meanwhile, prepare secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI) dilution in PBS.
   2. 100 µL of 1:2000 fluorophore-conjugated anti-Mouse IgG and 1 µg/mL DAPI will be required per well.
   3. NOTE: Dilution of secondary antibody used for IF may need optimizing if an alternative is used.
   4. Vortex mixture to ensure sufficient mixing.
8. Remove PBS from all wells and add 100 µL secondary antibody/DAPI dilution.
9. Incubate plate at room temperature for 1 h, protected from light.
10. Remove secondary antibody/DAPI from all wells.
11. Wash all wells with 200 µL PBS and remove.
12. Add 200 µL PBS to all wells and incubate for 5 min.
13. Remove PBS and add 100 µL PBS to all wells and store, protected from light, at 4 ^oC until ready to image.
    NOTE: Plates can be stored for up to one month with minimal loss in signal/image quality.

6. Imaging and counting of infected cells

1. To quantify infection easily and accurately, image at least 25% of each well, in both the blue (DAPI, nuclei) and secondary antibody (HCMV immediate-early proteins (IE)) channels.
   NOTE: The signal generated by the IE immunofluorescence should resemble that of the nuclear staining and images should be checked to confirm this.
2. This can be performed manually, but the use of an automated microscope is advised.
   NOTE: Magnification will depend on your instrument, but as large structures (nuclei) are being imaged, 4X/10X objectives are typically sufficient.
3. Once images are acquired, process them through the software analysis program of choice.
   1. First, identify nuclei via the blue channel, to calculate the number of cells present.
   2. Only these areas then need to be checked for signal in the red channel, as IE is a nuclear-localized protein, to calculate the number of infected cells.
4. Once the number of infected cells and the total number of cells are calculated, calculate the percentage of infection on a per well basis.
5. These values can be corrected for background levels of detection by subtracting the average percentage of infection observed in uninfected wells from all other wells.
   NOTE: This should be well below 1%. If it is more than 1%, the analysis procedure requires optimization.
6. These background-corrected values can now be plotted, interrogated, and analyzed as desired.