An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus
Published: February 29, 2024
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…
開示
The authors have nothing to disclose.
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 |
