4.4. Lentiviral Titer Calculation

This section describes how to calculate the titer of packaged lentiviral particles. To calculate the number of transducible viral particles in the viral stock, you need to know the number of cells transduced at a specific dilution factor of the stock virus. This can be assessed by counting RFP-positive cells on a flow cytometer or using staining to assess cells with antibiotic resistance (e.g., puromycin).

• It is important to use an amount of virus sufficient to only transduce a fraction of the target cells so that you can accurately assess the number of transductions occuring. You cannot accurately assess the number of transductions if the whole population is transduced (i.e., fluorescent, or die out due to antibiotic selection). Ideally, it is best to aim for transducing less than half the cells so that most of the cells have been transduced with a single viral particle. For this reason, it is typical to titer several dilutions of the viral stock to obtain a culture with enough transduced cells for the calculation but not to the point where there are more viral particles than cells.

• To calculate the titer of a viral stock, it is necessary to have the following information:
  • The number of cells at transduction.
  • The percent of transduced cells after transduction (i.e., the Transduction Efficiency).
  • The volume of the viral stock used to transduce the cells.

Calculate the Transduction Units at Infection.

Lentiviral titer is measured as Transduction Units per ml (TU/ml). One TU produces one integration event in target cells. To calculate the viral titer, it is first necessary to determine the number of Transduction Units (TU) used to infect the cells. When the percentage of infected cells is at or below 20%, the number of integrations is approximately equal to the number of transduced cells. However, at higher transduction levels, the fraction of transduced cells with multiple integrations increases, so that the percentage of transduced cells relative to integration events per cell is no longer linear. Using the chart below, the number of integrations per cell, or MOI (Multiplicity Of Infection), can be accurately estimated for cultures with up to 75% transduced cells (i.e., MOIs in the range of ~0.2-1.5).

Estimation of percentage of cells infected based on Multiplicity Of Infection (MOI).

Calculate the Stock Titer

To calculate the titer of the original viral stock, apply the formula below:

TU/ml = (# of cells at Transduction) × [MOI / (ml of Lentiviral Stock used at Transduction)]

• # of cells at Transduction = Total number of cells in the culture when viral particles were added
• MOI = Derived from the chart above based on the percentage of transduced cells.
• ml of Lentiviral Stock used for Transduction = The volume in ml of the virus added to the cells. Include any dilution of the viral stock.

Example Calculations:

IF:
The original # of cells at Transduction was 100,000, and
The volume of virus stock used was 10 μl, and
The observed % of transduced cells (RFP+ or antibiotic resistant) is 25%,

THEN:
The calculated MOI is 0.3 (from the chart), and

The TITER is:
100,000 × 0.3 / 0.01 = 3,000,000 TU/ml