Hi
I'm trying to transduce my CD3/CD28 bead activated purified T-cells.
I'm not having much success.
I've seen some people use MOI of as low as 1 with VSVG pseudotyped lentiviral vectors (LV). I've seen some protocols using polybrene ... for their transduction. But can agents such as this be used in clinical applications? If no, then how do they get appropriate transduction in clinic? A protocol from Miltenyi had suggested using Protamine sulphate. Has anyone any experience with that?
Also I've seen people mention Using 0.5 million cell/ml and using a low volume to increase the virus concentration (instead of just increasing MOI). What are your suggestions? For example in a miltenyi suggested protocol it was written:
count cells and place 450ul of cell suspension in single well of a 24-well plate at a density of approximately 1x106cells/ml.
b. Let cells settle, the carefully remove 150 ul from the top of each well.
c. To each well, add an appropriate dilution of lentiviral vector, diluted in in plain TexMACS medium, and protamine sulfate to a final concentration of 10 ug/ml, in a 150 ul volume for a final volume per well of 450 ul.
d. Incubate for 24 hrs.
Any thoughts on this?
cheers
mostafa
T cells are terrible to transduce with lenti virus. Retro virus is much better and can result in 80% transduction efficiency. If you can, switch to retro.
I'm trying to follow another protocol were they activate only using dynabeads at 3:1 ratio and some how got away with Lenti MOI of 1. I'm not sure what is their efficiency but they use the T-cells for cytotoxicity assays I assume should get more than 30% to have a valid experiment afterwards. I'm using a third generation vector with pMD2.G encoding VSV-G as the viral envelope.
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