An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

Abstract

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 Seed target cells the day prior to performing the experiment in a 96-well plate (the 'target' plate). 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. Typically, 21 wells (7 down by 3 across) should be seeded per sample, allowing 4…

Materials

Mouse monoclonal anti-IE antibody clone 6F8.2	Millipore	MAB8131	Primary antibody for immunofluoresence
Rabbit Anti-Mouse IgG, Alexa fluor 568 conjugated antibody	Invitrogen	A-11061	Secondary antibody for immunofluoresence
WiScan® Hermes High Content Imaging System	Idea Bio-medical	–	High throughput, automated fluorescent microscope for quantification
Metamorph Image Analysis software	Molecular Devices	–	Image analysis software
Human IgG1 Isotype Control	Merck	M5284	Control antibody for neutralisation
8F9, anti-HCMV glycoprotein B antibody	–	–	Neutralizing antibody for HCMV
DAPI	Thermo Scientific	62248	Staining of cellular nuclei
DMI4000B Inverted Fluorescence microscope	Leica	–	Fluorescent microscope used for representative images