Titration of Lentiviral Vectors

 

Day 1 (9-12am)

• plate 30.000 293t per well on 24 well plate in 1 ml medium/well

 

Day 2 (4-6pm)

• count cells on 1 well (should have 60-80.000)
• transduce the cells with 6, 4-fold serial dilutions in 250� total vol; take 50� for non-conc. or 1� 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. 1� gives you 10% of positivie cells and you had 50.000 cells on D2, so you have 5.000TU/� --> 5x10⁶ TU/ml

 

Day 7-8

• isolate DNA for QPCR