Overview
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 samples to be analyzed per plate.
- Seed an additional 12 wells per plate (i.e., row 8) to serve as controls.
2. Preparation of virus: antibody mixtures
- Add 90 µL of media combined with antibody (and well mixed) to create twice the desired initial test antibody concentration into 3 wells of row 1 of a fresh 96 well plate (the 'dilution' plate).
NOTE: A typical starting concentration of antibody would be 20 µg/mL but should be determined empirically. - Add 60 µL of media to each of the 18 wells below (i.e., 6 down by 3 across) the 3 wells from step 2.1.
- Add 60 µL of media to 12 additional wells (i.e., row 8).
- Transfer 30 µL of antibody dilution from row 1 to the corresponding wells in row 2.
- Mix the contents of row 2 by pipetting smoothly up and down 20 times with the multichannel pipette.
- Transfer 30 µL of antibody dilution from row 2 to row 3.
- Repeat this process of mixing and transferring until the contents of row 7 have been mixed.
- Once row 7 has been mixed, dispose of 30 µL from the wells in row 7.
- Dilute HCMV in a complete medium so that 50 µL contains sufficient infectious particles to result in a MOI=1 infection if added directly to the cells.
NOTE: HCMV is a Biosafety Level 2 (BSL-2) category pathogen. Ensure proper safety procedures are in place and use filter tips for all HCMV-exposed media.
NOTE: This requires the same number of infectious particles as there are cells/well, which will vary based on cell type and virus tropism. If using HFFs as described above, the required virus concentration is 4x105 pfu/mL.
NOTE: Ensure a sufficient excess of diluted virus (e.g., 10% extra) is prepared. - Add 60 µL of diluted virus stock to all wells containing antibody dilutions, and 6 wells that contain only 60 µL media.
- Add 60 µL of media only to the remaining 6 wells of media only, to serve as uninfected controls.
- Incubate the resulting virus: antibody mixtures at 37oC for 1 h.
3. Infection of target cells
- Remove all media from target cells.
- Using a multichannel pipette, transfer 100 µL of virus: antibody mixture from the ‘dilution’ plate onto the cells in the equivalent position on the 'target' plate.
- Incubate the cells at 37 °C, 5% CO2 for 24 h.
4. Fixation of cells
- Remove all media from cells.
NOTE: There is no need to change tips between wells at this point. - Wash all wells with 200 µL phosphate-buffered saline (PBS) and remove.
- Add 180 µL -20 oC, 100% ethanol to all wells.
- Incubate plate at -20 oC for at least 30 min.
NOTE: This step can be extended to at least one month, provided additional ethanol is added as it begins to evaporate.
NOTE: The plate is safe to handle under BSL-1 conditions from this point onwards, and non-filter tips can be used.
5. Detection of virally infected cells by immunofluorescence (IF)
- Remove ethanol from the plate.
- Wash all wells with 200 µL PBS and remove.
- Add 200 µL PBS to all wells and incubate for 5 min.
- Meanwhile, prepare primary antibody dilution in PBS.
- 100 µL of 1:2000 mouse monoclonal anti-IE will be required per well (note dilution is for the specific antibody listed in materials).
NOTE: Dilution of the antibody used for IF may need optimizing if an alternative is used. - Vortex mixture to ensure sufficient mixing.
- Remove PBS from all wells and add 100 µL primary antibody dilution.
- Incubate at room temperature for 1 h.
- Remove primary antibody from all wells.
- Add 200 µL PBS and incubate for 5 min.
- Meanwhile, prepare secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI) dilution in PBS.
- 100 µL of 1:2000 fluorophore-conjugated anti-Mouse IgG and 1 µg/mL DAPI will be required per well.
- NOTE: Dilution of secondary antibody used for IF may need optimizing if an alternative is used.
- Vortex mixture to ensure sufficient mixing.
- Remove PBS from all wells and add 100 µL secondary antibody/DAPI dilution.
- Incubate plate at room temperature for 1 h, protected from light.
- Remove secondary antibody/DAPI from all wells.
- Wash all wells with 200 µL PBS and remove.
- Add 200 µL PBS to all wells and incubate for 5 min.
- Remove PBS and add 100 µL PBS to all wells and store, protected from light, at 4 oC until ready to image.
NOTE: Plates can be stored for up to one month with minimal loss in signal/image quality.
6. Imaging and counting of infected cells
- To quantify infection easily and accurately, image at least 25% of each well, in both the blue (DAPI, nuclei) and secondary antibody (HCMV immediate-early proteins (IE)) channels.
NOTE: The signal generated by the IE immunofluorescence should resemble that of the nuclear staining and images should be checked to confirm this. - This can be performed manually, but the use of an automated microscope is advised.
NOTE: Magnification will depend on your instrument, but as large structures (nuclei) are being imaged, 4X/10X objectives are typically sufficient. - Once images are acquired, process them through the software analysis program of choice.
- First, identify nuclei via the blue channel, to calculate the number of cells present.
- Only these areas then need to be checked for signal in the red channel, as IE is a nuclear-localized protein, to calculate the number of infected cells.
- Once the number of infected cells and the total number of cells are calculated, calculate the percentage of infection on a per well basis.
- These values can be corrected for background levels of detection by subtracting the average percentage of infection observed in uninfected wells from all other wells.
NOTE: This should be well below 1%. If it is more than 1%, the analysis procedure requires optimization. - These background-corrected values can now be plotted, interrogated, and analyzed as desired.
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Materials
Name | Company | Catalog Number | Comments |
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 |