Hi! I am going to determine the functional lentivirus titers by transducing the virus into HEK-293T cells and then calculate the cell colonies expressing the GFP. Is it necessary to grow and fix the 293T cells on collagen I coated coverslips for this purpose? And does it going to make a big difference in transduction efficiency by adding polybrene in the transduction mixture? Can anyone give advice? Thanks.
No, you need not to grow 293T cells on collagen-coated coverslips unless you are using medium with low or no FBS. For 293T cell transduction, use of polypbrene looks a bit overkill to me, not to mention its toxicity that might kill a large number of cells. However, if you must use polypbrene, try various dilutions and pick the one least toxic towards cells. Better still, use either protamine sulfate or DEAE-dextran for these substances do enhance transduction and are not as toxic.
I agree with Syed that you do not need to grow the cells on collagen-coated coverslips.
Use of 8ug/ml final concentration of polybrene in the transduction mixture will not be toxic to the cells. I use this regularly when titering lentivirus and there is no cell death.
If you have access to a flow cytometer then you can get the titer of the virus by looking at the efficiency of transduction (via GFP expression) of the cells rather than counting colonies.
Good Luck!
Thank you Syed and Tom for the suggestions!
I had my first attempt without collagen coverslip and polybrene. Somehow I got very few green colonies even in 25 x dilution. My lentivirus particles were harvested from 2 mL of culture supernatant, concentrated by ultracentrifugation and resuspended in 500 uL of DMEM. The transfection for virus particles production went very well, about 90% of the cells were expressing GFP so I expect I would get high titer of virus. But it was not.
I think assess the efficiency of transduction using flow cytometer is a good idea as I also encounter the issue of counting green colonies, some of the colonies were green, but very dim, I was not sure if I should include them. I will give this a try and let you guys know how it goes later.
Thanks again.
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