Research and Development on Lentivectors for:

ADDGENEUNITEC

Dr Patrick SALMON @ Department of Neurosciences - Faculty of Medicine - University of Geneva

Lentiviral Vectors Lab @ UNIGE

Lentivector titration by FACS


2011GiryLaterriere MiMB.pdf

This method can only be used to titer stocks of vectors that carry a transgene that is easily monitored by FACS (such as GFP, or any living colors, or any membrane protein that can be detected by flow cytometry), and whose expression is governed by a promoter that is active in Hela cells (tissue-specific promoter-containing vector must be functionally assayed in specific cells, and titered by QPCR in Hela cells.

We describe here the titration of a PGK-GFP vector.


On day 0, seed Hela cells at 100kC per well in MW6 plate in DMEM 10% FCS, 1% glutamine and antibiotics.


On day 1, put in three independent wells 500, 50 or 5µl of the vector suspension (either pure from unconcentrated supernatants or diluted if it comes from a concentrated stock).


On day 2, remove the supernatant and replace by 2 ml of fresh D10.


On day 4 to 5, wash the cells with 2 ml of PBS, detach them with 250 µl of Trypsine-EDTA for 1 minute at 37C, add 250 µl of 2% formaldehyde in PBS (to fix the cells, inactivate the trypsin and the vector particles), resuspend thoroughly, and analyze them for GFP expression by FACS.


A reliable measure of the fraction of GFP+ cells relies on the level of GFP expression. In the example shown below, GFP-positive and GFP-negative cells can be readily discriminated when GFP is expressed from a human PGK promoter, and allowed to accumulate in cells for 4 days. A marker can then be set to measure the fraction of transduced versus total cells.









 

 


A representative FACS analysis of Hela cells used for titration of GFP-coding LV.


Hela cells (105) were incubated with various volumes of a supernatant containing a LV expressing GFP under the control of the human PGK promoter (pRRLSIN.cPPT.PGK.GFP.WPRE (Follenzi et al., 2000)). After 4 days, cells were detached, fixed and analyzed by FACS for GFP fluorescence (x axis, 4-decade log scale, FL1) versus number of cells (y axis, linear scale). The percentage of GFP-expressing cells was measured by placing a marker discriminating between GFP-negative (mean of fluorescence intensity 3-4) and GFP-positive cells (mean of fluorescence intensity 200).


In a typical titration experiment, only dilutions yielding to 1 to 20% of GFP-positive should be considered for titer calculations. Below 1%, the FACS may not be accurate enough to reliably determine the number of GFP-positive cells. Above 20%, the chance for each GFP-positive target cell to be transduced twice significantly increases, resulting in underestimation of the number of transducing particles. Once chosen the appropriate dilution, apply the following math:


Titer (Hela-transducing units / ml) = 100000 (target Hela cells) x (% of GFP-positive cells/100) / volume of supernatant (in ml).