Hi, I have some questions about my lentivirus.

1. When I measure the titer of virus, I use 2 fold diluted virus from 1:100 to 1:1600 and transduction the virus into HEK293FT cells (On 24 well plate, 1 X 10^5 cells / well).

But I can't get a linearity of my virus because there is no significant difference of the positive percentage between 1:100 and 1:200 fold diluted virus.

Does the 1:100 fold dilution has an effect on cell viability or morphology? And may I ask your dilution strategy for lentivirus titration?

2. How can I improve my lentivirus titer?

I produce 3rd generation lentivirus using HEK293FT cells grown O/N to approximately 70~80 % confluence in 100 pi dish (5 X 10^6 cells/plate). Cells were cultured in DMEM media with 10 % FBS and 1 % P/S.

And the day for DNA transfection, I change the media using DMEM with same components right before transfection.

And I transfect the DNA into cells using transIT2020 form takara. I use 7.5 ug/plate of transfer plasmid, 2.5 ug/plate of pMD2.G, pRSV-REV, pMDLg/RRE so the total DNA is 15 ug / plate.

I change my media after 6 h of transfection and collect the supernatant after 48 h.

Then I filter the supernatant with 0.45 um filter and harvest the virus pellet using Lenti-X concentrator from takara (1/3 volume of collected supernatant) and incubation O/N at 4 degree of celsius.

Then I collect the virus pellet by centrifugation 30 min, 3000 rpm and resuspension the virus pellet using suitable media (mTeSR1 and alpha-MEM and other media).

And my titer is about 2 ~ 3 X 10^5 / dish and is not enough to conduct further experiment.

So, are there any steps I should evaluate to enhance my titer?

Thank you for reading my questions and I hope get answers.