The most critical parameter when transducing cells in vitro with Integrase-Deficient Lentiviral Vectors (or episomal) is to optimize the contact between cells and vector particles. To achieve this, always favor transduction in small culture volume. Indeed, transduction of a given number of cells with 1µL vector in 1 mL cell culture medium will be far less efficient than with the same dose in 200 µL cell culture medium.

  •  When applicable, reducing the transduction volume allows to increase transduction efficiency without increasing the vector dose and possibly related toxicity.

The transduction volume is established according to several parameters:

  • The nature of cells, i.e. their sensitivity to transduction (see Protocol 1 and 2 for most robust cells, Protocol 3 for more sensitive cells)
  • The number of cells to transduce, i.e. the number of transduced cells needed for subsequent assay.

Find below 3 examples of protocol to help you to develop your own experimental protocols, keeping in mind the critical parameter: optimize contact between cells and lentiviral particles.

  •  The transduction is more efficient when cells are in good shape in their complete preferred culture medium so do not modify culture conditions during transduction. For instance, it’s not worth removing FBS.

Protocol 1: Standard transduction (infection) protocol

Cells / well (HEK293T)
Vector Quantity (TU)
Transduction volume
Growing volume
8.104 – 1.105
1.104 – 5.104
200 µl
500 µl
1.5 105 – 2.5 105
2.104 – 1.105
400 µl
800 µl
3.105 – 5.105
4.104 – 2.105
800 µl
2000 µl

Day 1

  •  Collect the required number of cells.
  •  Mix the determined dose of cells and vector and seed the transduction mix (cells + Lenti-ONE) in wells. Note that we favored 24-well plates or 12-well plate, if you need more cells for subsequent analyses; we suggest you to distribute your transduction mix in multiple wells.
  •  After 5-6 hours of contact in minimal volume, fill with fresh medium (up to 500µL or more in 24-well plates or 1mL or more in 12-well plates or 2mL or more in 6-well plates).

Day 2

  •  Replace medium.

  Note that some sensitive cells will not tolerate simultaneous seeding and transduction and will prefer seeding, adhesion and subsequent transduction (see below).

This protocol can be used in most cases, when cells are not too sensitive, or when the transgene does not exhibit low expression patterns. However, in those last cases, you might need to optimize the transduction to get a better efficiency. Two adapted versions of the standard protocol are available on the PDF version.

If you are having troubles getting suitable results in your experimental context, you can also contact us. We would be glad to help you with your issues and give you more insights on how to get the best of lentiviral vectors.