Nov 18, 2025
SeV titration using FFU
- Carolina Lopez1
- 1Washington University
Protocol Citation: Carolina Lopez 2025. SeV titration using FFU. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjxj5lx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 23, 2024
Last Modified: November 18, 2025
Protocol Integer ID: 100402
Keywords: ffu protocol for titration, sev titration, protocol for titration, using ffu protocol, titration, sev
Abstract
Protocol for titration of SeV
Materials
Infection medium
- DMEM …………………………. 500 mL
- Pen/Strep ………………………. 5 mL
- 35% BSA ……………………… 5 mL
- 5% NaHCO3 ………………….. 12 mL
2X Infection medium
- DMEM F12 (Powder) ………… 1 sachet
- Pen/Strep ………………………. 10 mL
- L/Glut ………………………….10 mL
- 35% BSA ………………………. 6 mL
- HEPES 1M ……………………… 10 mL
- NaHCO3 ……………………….. 18 mL
- Sterile dH2O …………………… to 500 mL
Overlay medium
- 2X Infection media
- 2.5% Sterile Avicel
- TPCK-Trypsin
Reagents for Staining
- Anti-SeV primary antibody (example: ms SeV NP antibody, Clone M73/2)
- Goat anti-mouse H+L Biotinylated Antibody (Southern Biotech #1036-08)
- Streptavidin AP (ThermoFisher #21324)
- 1-Step‱ NBT/BCIP Substrate Solution (ThermoFisher #34042)
Troubleshooting
Preparing Overlay Medium
To prepare the overlay medium, first, add the correct amount of TPCK to the 2X infection medium. Then mix the 2X infection media/TPCK with the same volume of 2.5% Avicel to prepare a 1:1 solution. Alternately, 0.6% Agarose can be used in a similar 1:1 manner for a final concentration of 0.3% Agarose. All media must be prepared in sterile conditions.
Example for 50 mL of Overlay medium using Avicel:
- 25 mL of 2x infection medium
- 50 µL of TPCK (2 mg/mL)
- 25 mL of 2.5% Avicel
Day 0 - SEEDING CELLS
- Seed 2x105 LLCMK-2 cells/well in a 24-well plate (or in a way they are 100% confluent next day).
- Suggestion:
- Trypsinize a whole, 80% confluent T75 flask;
- Resuspend cells in 10 mL (regular 10% DMEM) and seed 450 µL each well - Homogenize and check if cells are evenly distributed in the wells.
- If scaling up to 48-well plates, seed 105 cells/well.
- Incubate plates at 37 °C .
Day 1 - DILUTION AND INFECTION
1h
- Check cells and ensure they are 100% confluent.
- Prepare 10fold dilutions of your samples in Infection Media;
- Change tips for every dilution;
- Wash cells twice with PBS to remove any FBS trace;
- Inoculate 200 µL of your serially diluted samples on each well (if using 48-well plates inoculate 180 µL ).
- This step can be done in duplicates or triplicates.
- Inoculate in the opposite direction from the dilution so there is no need for changing tips;
- Save wells for the negative control (Infection media only) and the 2ry antibody negative control (Also infection media only).
- Incubate at 37 °C for 01:00:00 , gently rock the plate every 10';
- Remove the inoculum and add 500 µL of Overlay medium + TPCK 2 μg/mL (250 μL if using 48-well plates).
- Incubate the plates for 2 days at 37 °C .
1h
Day 3 (48hpi) - FIXATION
2h
- Aspirate the overlay medium from the wells and fix cells with 10% Formaldehyde (in PBS) for 02:00:00 . If using overlay medium containing Agarose, directly add 500uL 4% PFA over the wells and incubate as follows.
- 200 μL/well
- Use the plate rocker to avoid drying
- Remove the fixative agent and wash twice with PBS.
- Store at 4 °C or move directly to the staining steps.
2h
Day 3 - STAINING
1h
- Remove the PBS and replace with 150 µL of PBS/1% BSA 1%/0.1% Saponin.
- Incubate for 30' to 1hr to block/permeabilize cells
OBS.: All the following steps are done with 150 to 180 μL/well of reagent. Scale up according to the number of wells. To achieve better results also keep the plates on the rocker for all the following incubation steps.
- Aspirate the blocking/permeabilization solution and incubate with primary antibody;
- Unlabeled Ms anti-SeV NP (1.3 mg/mL) M73/2
- 1:2000 diluted in PBS/1% BSA 1%/0.1% Saponin;
OBS.: Don’t add the primary antibody to the 2ry-only negative control well.
- Incubate for 01:00:00 - rocking
- Wash 3x with PBS (180 µL of PBS/well, aspirate carefully for not scraping the monolayers).
- Incubate with secondary antibody for 30';
- Biotinylated Gt anti-Mouse (H+L) diluted 1:1000 in PBS/1% BSA 1%/0.1% Saponin.
- Wash 3x with PBS
- Incubate with Streptavidin for 30'
- AP-Conjugated Streptavidin, diluted 1:1000 in PBS/1% BSA 1%/0.1% Saponin.
- Wash 3x with PBS
- Incubate with AP substrate for up to 20' (or until the FFUs are pretty stained).
- 1-Step NBT/BCIP Substrate solution for AP
- Wash 1X with dH2O.
1h
Day 3 – FOCUS COUNTING
- In the last dilution with visible focus, count the stained focus in all the wells from that particular sample and divide by the number of replicates.
- The titer will be expressed by the average number of focus counted times the dilution factor, per volume of initial inoculum.
Figure 1. Representative titration plate. 10-fold dilutions of the initial virus inoculum are indicated in red. Infection media was used as negative control (NC), and is indicated in the bottom-right. Fixed NC monolayer was either incubated with the secondary (2ry) antibody only, or both primary and secondary antibodies as indicated.
- Example: In the plate above, there are 5 FFUs in the -7 dilution. Considering the dilution factor, the titer would be 5x107 FFUs. Finally considering the initial volume inoculated (200 uL), the final titer of that particular sample is 5x107 FFU/200uL.
Protocol references
Annotated by IAC