MOI calculator
How much virus to add for a target MOI, or the MOI you achieved. With the Poisson coverage.
How much virus to add for a target MOI, or the MOI you achieved. With the Poisson coverage.
The multiplicity of infection, or MOI, is the ratio of infectious virus particles to cells in an infection or transduction. An MOI of 5 means five infectious units were added for every cell in the well. It is the standard way to describe how hard cells were hit, because it is independent of the well size: the same MOI gives the same outcome in a 96-well plate and a T175 flask, provided the titre and the cell count are right.
Rearranged for the practical question, how much stock to pipette:
Worked example. One million cells are to be infected at MOI 5 from a stock titred at 1 × 10⁸ PFU/mL. The infection needs 5 × 10⁶ PFU, so the volume is 5 × 10⁶ ÷ 1 × 10⁸ = 0.05 mL, that is 50 µL of stock.
| Application | Typical MOI | Why |
|---|---|---|
| Plaque assay | ≪ 1 | Each plaque must come from a single particle |
| Lentiviral transduction | 0.5–10 | Low MOI favours single integrations; raise it for hard-to-transduce cells |
| Retroviral, single-copy | 0.1–0.3 | Keeps most transduced cells to one provirus |
| Adenovirus, transient expression | 10–100 | Non-integrating and often poorly infectious per particle |
| AAV | 10³–10⁶ VG/cell | Titred in genomes, most of which are not infectious |
| Synchronous infection for virus stocks | 5–10 | Every cell infected in the first round |
| Multi-cycle growth curve | 0.01–0.1 | Virus must spread through several rounds |
An MOI is only meaningful alongside the unit its titre was measured in. PFU, plaque forming units, counts particles able to form a plaque in a monolayer, measured by plaque assay.TU and IU, transducing and infectious units, count particles that deliver their genome to one cell, usually measured by flow cytometry or qPCR on transduced cells.VG, viral genomes, counts genome copies by qPCR whether or not the particle can infect anything. The particle-to-infectivity ratio of a typical preparation runs from ten to several thousand, so an MOI of 10 in VG may be an MOI of 0.01 in infectious units. Never compare an MOI across units, and always record which unit you used. For endpoint dilution titres, convert with the TCID50 calculator; for bacterial counts use theCFU calculator.
Virus particles land on cells at random, so the number of particles a given cell receives follows a Poisson distribution. If the mean number per cell is m, the MOI, then the probability of a cell receiving exactly k particles is:
The fraction of cells that receive nothing is e⁻ᵐ, so the fraction infected is 1 − e⁻ᵐ. At MOI 1 that is only 63%; at MOI 3, 95%; at MOI 5, 99.3%; and at MOI 10, 99.995%. This is why an MOI of 1 never transduces a whole culture, and why virus stocks are made at MOI 5 or more when every cell must be infected in the first round. The calculator reports these fractions for whatever MOI you enter. Two caveats: the model assumes cells are equally susceptible and that every infectious unit finds a cell, so real coverage is usually a little lower. Check what the infection did to the culture with thecell viability calculator.
The MOI depends on the number of cells at the moment of infection, not at seeding. A culture seeded the day before will have grown, often close to doubling, so using the seeding number can halve the real MOI. Count a parallel well on the day, or seed at a density you have already measured for that plate format and timepoint.
MOI is infectious particles divided by cells. To find how much virus to add, multiply the target MOI by the number of cells and divide by the titre of the stock: volume (mL) = MOI × cells ÷ titre (per mL). For 1 million cells at MOI 5 from a 1 × 10⁸ PFU/mL stock, that is 5 × 10⁶ ÷ 10⁸ = 0.05 mL, or 50 µL.
It depends on the aim. Lentiviral transduction usually runs at MOI 0.5 to 10, adenovirus at 10 to 100, and a synchronous infection for virus production at 5 to 10 so that every cell is hit at once. A plaque assay needs MOI well below 1 so that each plaque comes from a single particle. Selectable transductions are often kept below MOI 1 so most positive cells carry a single integration.
Because particles distribute randomly. The Poisson distribution says the fraction of cells receiving no particle is e^−MOI, so at MOI 1 about 37% of cells are missed and only 63% are infected. Reaching 99% requires MOI 5, and 99.99% requires MOI 10.
They count different things. PFU counts particles that form a plaque, TU and IU count particles that transduce or infect one cell, and VG counts viral genomes whether or not they are infectious. A prep can have a hundred or a thousand times more genomes than infectious units, so an MOI based on VG is not comparable to one based on PFU or TU.
At infection. Cells divide between seeding and infection, often doubling in 24 hours, so using the seeding number can halve your effective MOI. Count a parallel well on the day of infection when the MOI matters.