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.
1. Preparation of target cells for infection
2. Preparation of virus: antibody mixtures
3. Infection of target cells
4. Fixation of cells
5. Detection of virally infected cells by immunofluorescence (IF)
6. Imaging and counting of infected cells
The authors have nothing to disclose.
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 |