An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

On the day before the experiment, prepare target cells for infection by culturing fibroblast cells, grown in complete media, consisting of DMEM supplemented with 10% FBS, in a sterile 96-well plate.

After 24 hours, prepare the virus-antibody mixture by adding 90 microliters of diluted antibody solution into the wells of a fresh 96-well plate. Prepare serial dilutions of antibody solution using complete media.

Additionally, add 60 microliters of the virus solution, prediluted in complete media, into each antibody-containing well to generate various concentrations of the virus-antibody mixtures and incubate.

Now, to infect the target cells, take the plate seeded with fibroblasts. Add 100 microliters of different concentrations of the virus-antibody mixture to the cells, and incubate the target plate for 24 hours.

After 24 hours, to detect the viral infection using immunofluorescence, fix the cells with absolute ethanol and incubate them in -20 degrees Celsius freezer. Post-fixation, proceed with the subsequent steps on the laboratory bench.

Pipette 100 µl of the primary antibody to all the wells of the plate. Place the plate at room temperature for an hour.

Remove the primary antibody solution from the wells. Add a mixture of fluorophore-conjugated secondary antibody and DAPI solution to all the wells. Incubate the plate for an hour in the dark, and wash the plate.

To quantify infection, visualize the plate under a microscope, and acquire images using the blue and red channels. Calculate the percentage of infection per well, as detailed in the text protocol.