An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

Abstract

Source:
Matthew J Murray¹, Gary R McLean^2,3, Matthew B Reeves<sup>1

1</sup>Institute of Immunity and Transplantation, University College London.
²Cellular and Molecular Immunology Research Centre, London Metropolitan University.
³National Heart and Lung Institute, Imperial College London.

This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence.

Protocol

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.