Titration of Lentiviral Vectors

Day 1 (9-12am)

Plate 30’000 293T cells per well on 24 well plate in 1 ml medium/well

Day 2 (4-6pm)

Count cells in 1 well (should have 60000-80000)

Transduce the cells with 6, 4-fold serial dilutions in 250µl total volume; take 50µl for non-conc. or 1µl for conc. vector stock for as a first dilution

Day 3 (9-12am)

Add 1ml of medium

Day 5

Split cells and analyze fluorescence by FACS and read percentage from linear values (usually 5-10%) Titer is a number (percentage) of cells transduced by a given vol and counted on D2 e.g. 1µl gives you 10% of positivie cells and you had 50.000 cells on D2, so you have 5.000TU/µl --> 5x106 TU/ml

Indeed, you can calculate the titer in transducing units (TU)/ml, according to the formula: (Number of the cells at the day of transduction×%GFP positive cells)/Volume of Vector (ml)

For accurate titer calculations, the number of GFP-positive cells in 2 wells infected with 2 consecutive dilutions must be close to the expected 1:2 ratio. This linearity is observed when <15% of the target cells are transduced.

Day 7-8

Isolate DNA for real time PCR (if titration by real time PCR is aimed to be done)

Adapted from Torno Lab Protocols

For more information on Lentiviral vector titration methods refer to:

Sastry et al. Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther. 2002 Sep;9(17):1155-62.

Reference: Current Protocols in Human Genetics, Unit 12.10, Production of High-Titer Lentiviral Vectors