LCMV Focus-Forming Assay (Immunofocus)

Dillon Corvino

Quantification of infectious titer (FFU/mL) of LCMV using antibody-based detection of infected foci on Vero cells with semi-solid overlay

Protocol ID: MUS-LCMV-FFA
Version: v1.2
Author: Dillon Corvino

Purpose

This protocol describes a focus-forming assay (FFA) for determining infectious titers of LCMV stocks using antibody-based detection of infected foci on Vero cells with a semi-solid overlay.

Compared with a classic plaque assay, the FFA:

• Uses immunostaining (e.g. anti-NP, VL-4) to visualize infected foci.
• Can detect infection earlier (typically 48–72 h post infection).
• Is useful for strains or conditions with limited cytopathic effect and for titrating experimental virus stocks.

WarningLCMV biosafety and handling

LCMV is a zoonotic arenavirus capable of causing severe disease in humans, including meningitis and congenital infection. Work with live LCMV must:

• Be conducted under the appropriate institutional biosafety level (typically BSL-2 with additional local conditions) and approved animal and biosafety protocols.
• Use dedicated infectious CO₂ incubators and clearly labelled containment for all cultures and waste.
• Include suitable PPE (lab coat/gown, gloves, eye protection as appropriate) and measures to avoid sharps injuries, aerosol formation, and spills.
• Exclude or take special precautions for pregnant or immunocompromised personnel according to institutional policies.

All waste (liquid and solid) must be inactivated according to local regulations (e.g. chemical disinfection and/or autoclaving) before disposal.

Approximate timing

• Vero maintenance and expansion: ongoing (split every 2–3 days as needed)
• Day −1: Vero seeding for infection (default method): 30–60 min, depending on number of plates
• Day 0: Virus dilution and infection, overlay addition: 1.5–2.5 h, depending on number of samples and dilutions
• Day 2–3: Fixation and immunostaining: 3–4 h hands-on time (spread over incubation steps)
• Focus counting and titer calculation: 30–60 min per plate, depending on number of foci

Total calendar time: ~2–3 days from infection to readout, plus routine cell culture maintenance.

Principle

• Serial 5-fold dilutions of virus are prepared in a 24-well dilution plate and used to infect a suspension of Vero cells in a matching 24-well incubation plate.
• After a short incubation (adsorption/infection phase), cells are overlaid with a semi-solid methylcellulose/2× DMEM mix and incubated for 60–72 h to allow local spread and formation of discrete foci.
• Cells are fixed, permeabilized, and stained with primary anti-LCMV antibody (VL-4, rat anti-NP), followed by an HRP-conjugated goat anti-rat IgG secondary antibody.
• Foci are visualized using an OPD (o-phenylenediamine) substrate and counted under a microscope.
• FFU/mL is calculated from focus counts, dilution factor, and inoculum volume.

Materials

Cells and Media

• Vero cells (African green monkey kidney)
• Vero propagation medium (see buf_vero_propagation_medium.qmd for detailed recipe)
  • Essentially (DMEM + 10% FCS + 2-mercaptoethanol)
  • Always use virus-titre-tested FCS
• Trypsin-EDTA for passaging Vero cells
• PBS without Ca^2+/Mg2+
• Infection/dilution medium (e.g. DMEM + 10% FCS + 2% Pen/Strep) for virus dilutions

Overlay and Related Buffers

• 2% methylcellulose solution (w/v) in dH₂O, sterilized (e.g. autoclaved)
• 2× DMEM overlay medium, sterile
• Optional: pre-warmed PBS for washing

Virus

• LCMV stocks (e.g. Armstrong, Clone 13, WE, Docile) stored at −80°C in single-use aliquots

Antibodies and Detection Reagents

• Primary antibody:
  • VL-4 (rat anti-LCMV NP), stored and handled according to supplier or in-house conditions
• Secondary antibody:
  • Peroxidase AffiniPure Goat Anti-Rat IgG (HRP-conjugated), or equivalent
• Chromogenic substrate:
  • OPD (o-phenylenediamine) tablets or powder for HRP-based detection, with appropriate buffer (e.g. citrate-phosphate) and H₂O₂

Fixation and Permeabilization

• 4% paraformaldehyde (PFA) in PBS
• Permeabilization buffer:
  • PBS + Triton X-100 (final concentration e.g. 0.1–0.3%)
• Blocking/antibody diluent buffers:
  • 5% FCS in PBS
  • 2% FCS in PBS (for primary and secondary antibody dilutions)

General Reagents and Consumables

• Sterile 24-well tissue culture plates:
  • One or more incubation plates for Vero cells
  • Matching dilution plates for virus serial dilutions
• Sterile conical tubes (15 mL, 50 mL)
• Microcentrifuge tubes
• Micropipettes and sterile filtered tips
• Serological pipettes (e.g. 5 mL, 10 mL, 25 mL)
• Waste containers with freshly prepared disinfectant (e.g. 10% bleach)

Equipment

• Class II biosafety cabinet
• Dedicated infectious CO₂ incubator set to 37°C, 5% CO₂
• Inverted light microscope
• Humidified chamber for antibody incubations
• Timer
• Fume hood for OPD preparation and development

Warning

Always use a designated infectious incubator for LCMV work. Do not use the same CO₂ incubator for sterile cell culture expansion and virus-infected plates.

Preparation

Important

Always use virus-titre-tested FCS

Vero cell maintenance

• Maintain Vero cells in Vero propagation medium (see buf_vero_propagation_medium.qmd) and passage every 2–3 days when cells reach ~60–80% confluence.
• For routine splitting:
  • Aspirate medium.
  • Wash with ~10 mL PBS.
  • Add 5–7 mL trypsin-EDTA and incubate at 37°C for 5 min, monitoring under the microscope until cells detach.
  • Neutralize trypsin with 20–30 mL Vero propagation medium.
  • Collect cells into a 50 mL tube and centrifuge at 20°C, 10 min, 400-500g.
  • Aspirate supernatant and resuspend the pellet in an appropriate volume (e.g. 5–10 mL) of fresh medium.
  • Seed into flasks/plates at the desired density for continued expansion.

Cell seeding for FFA

Two seeding strategies are described below. The default method seeds Vero cells the day before infection to form an adherent monolayer. The alternative method seeds Vero cells as a suspension immediately before infection, as described in the collaborator protocol.

Default method (recommended): Day −1 monolayer seeding

1. One day before infection (Day −1), prepare a single-cell suspension of Vero cells from a near-confluent flask.
2. Count cells using a hemocytometer or automated cell counter.
3. Dilute cells in Vero propagation medium to reach 70–90% confluence in 24-well plates on the day of infection (Day 0). As a starting point:
   • Aim for ~1–2 × 10^5 cells per well in 24-well plates.
4. Seed cells in 24-well incubation plates (e.g. 0.5–1 mL per well).
5. Incubate overnight at 37°C, 5% CO₂ in the infectious incubator.

Alternative method: Same-day seeding and infection (suspension-based)

This method is based on the collaborator’s protocol and may be useful when a tightly controlled cell number per well is desired.

1. On Day 0, detach Vero cells from a near-confluent flask as described under maintenance.
2. Resuspend cells in Vero propagation medium and count.
3. Calculate total cell number in the suspension. For example, if a counted sample corresponds to N cells per defined volume, determine the concentration and total cells in the resuspension volume.
4. Dilute the cell suspension to 8 × 10^5 cells/mL in Vero propagation medium.
5. Keep the cell suspension on ice until seeding.
6. For each well of the 24-well incubation plate, add 200 µL of this suspension (→ 1.6 × 10^5 cells per well).
7. Proceed directly with virus addition and infection as described in the infection section.

Tip

The monolayer (Day −1) method may provide slightly more uniform adherence, while the same-day suspension method offers tightly controlled cell numbers per well. Choose one approach and use it consistently within a given experiment.

Overlay media

2% methylcellulose

• Prepare a 2% (w/v) methylcellulose solution by dissolving 20 g methylcellulose in 1 L dH₂O.
• Autoclave (if compatible with your methylcellulose product) or sterile-filter if possible.
• Store at Room Temperature.
• Place on a magnetic stirrer to mix thoroughly before combining with 2× DMEM.
• Do not invert or vortex vigorously, as this can introduce bubbles.

2× DMEM overlay medium

Prepare 2× DMEM overlay medium using 10× DMEM base lacking L-glutamine, sodium pyruvate, and NaHCO₃, and supplement with the following components:

• 10× DMEM base (no L-glutamine, no sodium pyruvate, no NaHCO₃)
• Sodium pyruvate (100 mM stock)
• NaHCO₃ (74 g/L stock solution)
• L-glutamine (200 mM stock)
• Penicillin/Streptomycin (100× stock)
• FCS (use virus-titre-tested FCS)
• Bidest water (to adjust to final volume)

Adjust volumes according to the desired final preparation volume and concentrations (see table below).

Final concentrations

The overlay is later mixed 1:1 with 2% methylcellulose, so all concentrations halve in the final 1× overlay.

Component	Stock	In 25 mL 2× DMEM	Final in 2× DMEM	Final in 1× overlay
DMEM base	10×	5.00 mL	2×	1×
Sodium pyruvate	100 mM	0.50 mL	2 mM	1 mM
NaHCO₃	74 g/L (880.84 mM)	4.94 mL	14.62 g/L (174 mM)	7.3 g/L (86.89 mM)
L-glutamine	200 mM	0.50 mL	4 mM	2 mM
Pen/Strep	100×	0.50 mL	2× (2% v/v)	1× (1% v/v)
FCS	—	2.50 mL	10% (v/v)	5% (v/v)

Mix thoroughly. Store at 4 °C and warm to room temperature or 37 °C before use.

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Overlay mixture

The overlay is prepared as a nominal 1:1 mix of 2× DMEM and 2% methylcellulose, yielding a final 1× DMEM, 1% methylcellulose overlay.

Because methylcellulose is viscous and adheres to vessel walls, prepare with ~10% excess methylcellulose to ensure correct final volume.

Example preparation

• 25 mL 2× DMEM overlay medium
• 27.5 mL 2% methylcellulose

Vigorously shake to homogenize, then place in a 37 °C water bath to remove bubbles and equilibrate before use.

Tip

Because the methylcellulose overlay is viscous, use a cut pipette tip (trimmed with sterile scissors) or a serological pipette to avoid bubbles and ensure accurate dispensing. Note: it can also be pipetted normally with a 200ul or 1,000ul pipette tip

Virus dilution series (5-fold plate-based)

Note

Perform titration in duplicate

1. Thaw a virus aliquot Standard here = on ice slowly Other published protocols = rapidly at 37°C and immediately transfer to ice.
2. Prepare a 24-well dilution plate:
   • Add 400 µL infection/dilution medium (e.g. DMEM + 10% FCS + 1% Pen/Srep) to each well required for the serial dilution.
3. For each virus stock:
   • Add 100 µL virus stock to the first well in the column (top row) to obtain a 1:5 dilution.
   • Mix by pipetting up and down gently.
4. Serial 5-fold dilutions:
   • Transfer 100 µL from the first well to the next well in the column containing 400 µL medium.
   • Mix thoroughly by pipetting.
   • Repeat down the column to achieve the desired range of 5-fold dilutions (e.g. dilutions 5^1 to 5^5 or more, depending on expected titer).
5. Use fresh tips for each transfer to avoid back-contamination.
6. Include a no-virus control
7. Keep the dilution plate on ice until infection.

Infection and incubation

1. Ensure Vero cells are prepared according to your chosen seeding method (monolayer or same-day suspension) in the 24-well incubation plate.
2. For each virus dilution:
   • Transfer 200 µL from the corresponding well of the dilution plate onto the appropriate well of the incubation plate.
   • Each infection well should now contain:
     • 200 µL Vero cell suspension (if using same-day method) or the monolayer in existing medium (if using Day −1 method; in that case, aspirate most of the medium first, leaving a thin layer to prevent drying).
     • 200 µL of virus dilution.
3. Gently rock the plate to distribute the inoculum evenly.
4. Incubate at 37°C, 5% CO₂ in the infectious incubator for 3 h, gently mixing in a figure-eight pattern every 15 mins (adsorption/infection phase).
5. After 3 h, without aspirating the inoculum, overlay each well with 400 µL of the pre-mixed methylcellulose/2× DMEM overlay (see above).
   • Use a cut pipette tip or serological pipette to handle the viscous overlay and avoid bubbles.
6. Return the plates to the infectious incubator and incubate at 37°C, 5% CO₂ for 48–72 h. N.B Anna finds better foci at 48 hour incubation
   • Do not disturb the plates during this period to allow discrete foci to form.

Tip

A typical schedule is Day 0: infection and overlay, Day 2–3: fixation and staining. For example, Monday infection → Wednesday or Thursday readout.

Fixation

From this point on, all steps are performed at room temperature (unless otherwise specified). Work in the biosafety cabinet until fixation is complete.

1. After 48–72 h incubation, remove plates from the incubator and place them in the biosafety cabinet.
2. Carefully aspirate the overlay and supernatant without touching the cell layer.
3. Add 300–400 µL of 4% PFA in PBS to each well.
4. Incubate for 30 min at room temperature.
5. Aspirate the fixative into a container containing an appropriate disinfectant (e.g. 10% bleach).
6. Gently wash wells 2–3× with PBS.

Once fixation is complete and the plates have been washed, subsequent steps (permeabilization, antibody staining, OPD development) can be performed outside the BSL-2 cabinet if permitted by local biosafety regulations, but OPD handling must be done in a fume hood.

Permeabilization and priming

1. Add 200 µL of 1% Triton X-100 permeabilization solution (Diluted in PBS) to each well.
2. Incubate for 20 min at room temperature.
3. Aspirate the permeabilization solution.
4. Add 400 µL of 5% FCS in PBS to each well.
5. Incubate for ~20 min at room temperature.
6. Aspirate the 5% FCS/PBS solution immediately before adding primary antibody.

Antibody staining

Primary antibody (VL-4)

1. Prepare the primary antibody solution fresh on the day of use:
   • Dilute VL-4 (rat anti-LCMV NP) 1:400 in 2% FCS in PBS.
   • location: VL-4 antibody is found in Paula’s personal fridge, DO NOT USE THE OLD VIAL
2. Add 200 µL of the VL-4 working solution to each well.
3. Incubate for 60 min at room temperature covered to prevent evaporation.
4. After incubation, wash wells 2× with 300 µL PBS.

Secondary antibody (HRP-conjugated goat anti-rat IgG)

1. Prepare the secondary antibody solution fresh on the day of use:
   • Dilute anti-rat IgG-POX 1:100 in 2% FCS in PBS.
   • Location: Paula’s -20 freezer.
2. Add 200 µL of the secondary antibody solution to each well.
3. Incubate for 60 min at room temperature covered to prevent evaporation.
4. Around 10 min before the end of the secondary incubation, prepare the OPD substrate solution (see below).
5. After incubation, wash wells 2× with 300 µL PBS.

OPD development and stopping the reaction

Warning

OPD is hazardous and must be handled in a fume hood with appropriate PPE. OPD solutions and contaminated consumables must be collected as chemical hazardous waste according to institutional regulations.

1. Prepare the OPD substrate solution fresh approximately 10 min before use. Perform under fume hood

• Dissolve 1 tablet (20 mg) in 50 ml of 0.05M Phospho Citrate buffer
• Add 20 µL of 30% H2O2 (Add just before use)

2. Transfer the fixed, antibody-stained plates to a fume hood.
3. Add 400 µL of OPD substrate solution to each well.
4. Incubate for 10–20 min at room temperature, monitoring the development of foci periodically.
   • Stop the reaction once plaques/foci are clearly visible with good contrast but before background staining becomes excessive.
5. When development is complete:
   • Aspirate OPD solution into a designated chemical waste container in the fume hood.
6. Wash wells 2× with 400 µL PBS to remove residual substrate.
7. Air-dry plates in fume hood for ~24 hours.

Focus counting and titer calculation

1. Examine wells under a light microscope at low magnification (e.g. 4× or 10× objective).
2. Identify wells and dilutions that have 10–100 discrete foci (well-separated, countable).
3. For each suitable well, record:
   • Dilution factor (e.g. 5^3 = 125)
   • Number of foci
   • Volume of virus inoculum added per well (in mL; e.g. 0.2 mL from the dilution plate)
4. Calculate focus-forming units per mL (FFU/mL)

Formula:
FFU/mL = (Number of foci × Dilution factor) / Volume of inoculum (mL)

Example (5-fold dilution):

• 40 foci in a well
  
• Inoculated with 0.2 mL of a 5^4 dilution (5^4 = 625)

FFU/mL = (40 × 625) / 0.2 = 125,000 FFU/mL = 1.25 × 10^5 FFU/mL

5. If multiple wells at the same dilution are counted (technical replicates), calculate the mean FFU/mL and optionally the standard deviation.
6. Report titers as FFU/mL and record all raw counts and calculations in your lab notebook or electronic record.

Controls and quality checks

• Mock-infected wells (no virus) to assess background staining and non-specific antibody binding.
• Positive control virus stock of known titer to monitor assay performance across experiments.
• Wells should exhibit:
  • Minimal background staining in mock-infected controls.
  • Clear, discrete foci in infected wells.
• If background is high or foci are poorly defined:
  • Check permeabilization and blocking conditions.
  • Optimize primary and secondary antibody dilutions.
  • Confirm that overlay viscosity and incubation time are appropriate.

Buffers Required

Full composition, preparation, storage, and safety information for each buffer lives in its own linked buffer document — only a concise per-buffer summary is reproduced here, generated automatically from that document.

Vero Cell Propagation Medium

Component	Final concentration / amount
DMEM (high glucose, with L-glutamine, phenol red)	to 500 mL
Heat-inactivated FCS/FBS	50 mL (10% v/v)
2-mercaptoethanol (100x stock)	0.5 mL (1x final, typical 50-100 uM range)

• Final volume: 500 mL
• Preparation note: Mix gently by inversion; sterile-filter (0.22 um) if any component was added from a non-sterile container.
• Storage: 4C, protected from light, up to 4 weeks
• Caveat: Always use DMEM-based medium for Vero cells - do not confuse with RPMI-based media used elsewhere in the plaque/FFA protocol.

Full composition, preparation steps, storage, and safety information: BUF-VERO-PROP.

Troubleshooting

Issue	Possible cause	Suggested action
No foci at any dilution	Virus inactive or dilutions too high	Test lower dilutions; verify virus storage and freeze–thaw history
Very high foci numbers in all wells	Virus titer underestimated; dilutions too low	Increase dilution range (more 5-fold steps)
High background staining	Inadequate blocking or washing; antibody too concentrated	Increase blocking time; add more/longer washes; titrate antibodies
Foci too small or faint	Short incubation; suboptimal antibody concentration	Extend incubation (up to 72 h); increase primary or secondary slightly
Foci merging into large patches	Incubation too long; inoculum too concentrated	Shorten incubation; use higher (more dilute) dilutions
Uneven staining or edge effects	Incomplete coverage during antibody or OPD steps	Ensure sufficient volume and gentle rocking; avoid plate drying
Bubbles or holes in overlay	Viscous overlay handled with narrow tips	Use cut tips or serological pipettes; avoid vigorous pipetting

Safety considerations

• Confirm that all work with LCMV and infected animals/samples is covered by approved biosafety and animal use protocols.
• Perform all live-virus manipulations (thawing virus stocks, infection steps, handling of unfixed plates) in a certified Class II biosafety cabinet.
• Use dedicated infectious incubators and clearly label plates, flasks, and waste containers with the virus strain and date.
• Wear appropriate PPE at all times (lab coat/gown, gloves, eye/face protection as required).
• Decontaminate all liquid waste (e.g. with freshly prepared bleach) before disposal. Solid waste (tips, plates, tubes) must be autoclaved or otherwise inactivated according to institutional guidelines.
• OPD and its solutions are handled as chemical hazardous waste; follow chemical safety rules in addition to biosafety rules.
• Pregnant or immunocompromised personnel should not work with LCMV unless explicitly allowed under institutional policy with additional safeguards.

Version history

Version	Date	Description
v1.0	2025-12-01	Initial LCMV focus-forming assay (FFA) SOP (generic, MC57G/Vero, DAB-based).
v1.1	2025-12-02	Updated to Vero-only FFA with methylcellulose overlay, VL-4/OPD staining, and revised timing.
v1.2	2026-09-18	Dillon Corvino - Replaced # Buffers used with an auto-generated # Buffers Required section (one heading + one small table per buffer, sourced from each buffer’s own summary: data), moved to the correct position after Procedure, and fixed leaked R package-startup warnings.