I used to grow my undifferentiated LUHMES cells (dopaminergic neurons precursors) in 75 cm2 bottles and then add differentiation medium for 24hrs and seed them into four 21 Cm2 bottles and grow for 5 days before my experiments. I would like to transduce them with lentiviral particles and achieve the best efficiency without significant adverse effects such as toxicity. I heard that Polybryne is toxic to neurons. What additive would be better? I have read that fibronectin enhance lentiviral attachment. As I coat my cell culture plastics with fibronectin I thought it may be enough to add lentivirus to the coated bottle before seeding the cells and then seed them directly "on" the lentiviruses - has someone tried this? Do you need to get rid of the spare lentiviral sollution int the bottle before seeding to achieve good attachment? Will this not interfere with cell attachment in general? Is it better to transduce already attached cells and reseed them the next day post transduction? Is it possible to achieve good efficiency without any additives? Please share your validated protocols for LUHMES or other neuronal cells lentiviral transduction.
I would suggest to use the modified fibronectin, which binds better to the virus. I used retronectin, but I do not work with neural cells. However, I did not achieve very good results with it.
Instead, I used for mesenchymal stem cells viroductin. It worked good, but it is quite expensive. Instead of that I would recommend DEAE-dextran supplement with Lentiviral enhancer from ABM good.
I hope that I helped you somehow!
Hi
In our experience, polybryne never worked as it is said. You just have to use vectors
with VSVG and also higher titers of virus. I hope your insert is not that big, because in
that case getting good virus titer is not very easy. If you are having problems with
getting good titer of virus, try using a lot higher concentration of Packaging plasmid
(like twice or three times of what is recommended). I know what I am saying.
One more thing, try not to use huge number of the cells for transduction (24-well plate
is good) and also, try transduce cells before 24 hours post seeding the cells. If all the
thing here I mentioned didnt work, then you go try other things.
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