Hi, Chiara,

I think you have added too much virus particles. Here is my protocol of lentivirus titration.

a. Seed 293T 1×10^4 cells/well in a 96-well plate one day in advance.

b. Perform gradient dilution of the lentiviral particles to 1:10, 1:100, 1:1000, 1:10^4, 1:10^5, 1:10^6 in 100ul final volume in culture medium.

c. Total 100ul viral particle mixture should be added to each well with at least 3 replicates per virus.

d. Two days post infection, count the fluorescent positive cells using fluorescent microscopy and select the dilution factor with a proper fluorescent positive proportion (10%-30% positive cells/well). Count the triplicates and average the number of positive cells.

e. Estimate the lentivirus titer using the following formulation: Viral titer (TU/ml) = number of fluorescent positive cells × 10 × dilution.

Genemedi got a rich experience in lentivirus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging.

Hope this may help you.

Hi Chiara. If you see almost 100% of GFP positive cells you can't calculate your titer from that, because your cells might be infected with many viruses and that will bias your calculation. You should do serial dilutions of your viral stock, label your nuclei with DAPI, under microscope chose dilution which gives you single infected cells (20-60%) and count GFP to DAPI positive cells. Count few fields of view and average your result. Now knowing how many cells you have in your well calculate how many viruses are there and then knowing your virus stock dilution calculate what's the titer. 

From data you have Now, you can only say that your 10ul of viral stock infected 250000 cells so in 1ml you have MORE than 2.5 x 10e7.

as Anna said, you have added too much stock for the titre. her answer is correct, but if you do this often you may wish to use flow cytometry as it is faster, more accurate (you will likely miss weak GFP+ by microscopy) and doesn't make you feel sick staring down a microscope counting cells that keep going out-of-focus every time you change the field of view. 

Do 10-fold serial dilutions (as a guide, unconcentrated lentivirus is normally about 10^7 per ml). Count the number of cells at the time of transduction. Once confluent, collect cells and use flow cytometry to get the % of GFP+ cells. You want the dilution that gives you between 1 and 10% GFP+. multiple the % gfp+ by the number of cells at the time of transduction (when you added vector) to get the number of transducing units in the volume you added. adjust to get transducing units per ml.

e.g. if 0.1ul vector added to 50 000 cells gave 2% GFP+, you had 1000 (1e3) transducing units in 0.1ul. thats 1e7 per ml

unless you concentrated your supernatant, a good dilution series to start with is 1ul, 0.1ul, 0.01ul on 50 000 cells seeded in a 12 well plate. at confluent that will provide more than enough cells to get a % by flow cytometry.

Hi Chiara! To add to the previous answers - if you quantify just number or % of infected (EGFP+) cells, you will not be able calculate the titer in terms of "number of infectious viral particles in ul/ml". The lentivirus is integrative, and it will label not only the host cell it infects, but also the progeny of the cell. So, one viral particle could eventually label 20 cells if the original cell and its progeny divided several times. In this regard, it is easier to quantify viral tiger using adherent cells, like HEK293, which are a common cell line for viral titer quantification. If you could do it with adherent cells, you should infect cells by serial dilutions of your virus stock (as suggested above) in a minimal amount of cell culture media at 50-60% confluency and wait several days (we usually wait 3 days). Then you quantify not number of cells, but number of colonies. You will see that it could be 1-cell colony, but also could be 20-cell colony. Thus, you should use a dilution when the colonies are sparse. We usually do it in 96-well plates and good dilution for quantification would be between 10 and 30 colonies. Of course, it is better to use 2-3 wells for each dilution in order to quantify the titer precisely. However, infection rate could vary between cell types and tissues (depending on availability of receptor for lentiviral infection). HEK cells are just a common cell type to quantify the titer. So, the number you will get will be actually "infectious viral particles/ml for HEK cells", which could be different e.g. if you inject viruses in vivo.

Konstantin is right for microscopy method, but my flow cytometry method allows for cell division because you calculate based on the number of cells at transduction, not number at analysis. e.g., if 10 000 cells at time of transduction, 1% GFP+ at flow, then there were 100 transducing units. It is irrelevant that there might be 400 GFP+ cells due to 2 population doublings between transduction and analysis.

The assumption for above is that transduced cells divide at the same rate as non-transduced. But most adherent cells can migrate, so colonies isn't perfect either.

Hi, Chiara,

I think you have added too much virus particles. Here is my protocol of lentivirus titration.

a. Seed 293T 1×10^4 cells/well in a 96-well plate one day in advance.

b. Perform gradient dilution of the lentiviral particles to 1:10, 1:100, 1:1000, 1:10^4, 1:10^5, 1:10^6 in 100ul final volume in culture medium.

c. Total 100ul viral particle mixture should be added to each well with at least 3 replicates per virus.

d. Two days post infection, count the fluorescent positive cells using fluorescent microscopy and select the dilution factor with a proper fluorescent positive proportion (10%-30% positive cells/well). Count the triplicates and average the number of positive cells.

e. Estimate the lentivirus titer using the following formulation: Viral titer (TU/ml) = number of fluorescent positive cells × 10 × dilution.

Genemedi got a rich experience in lentivirus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging.

Hope this may help you.

Hi Chiara, you will find the exact title in this protocol of lentivirus, hope it can help you.