How can I transfer transfect Culture of hippocampal neurons with GFP?
the more I read I get confused
correct me if I am wrong according to my understanding.
1) the neuron need to extract
2) then culture or transfect using for example Lipofectamine 3000
please if you know any articles that help me to understand share it with me
thank you
Hi Ryan. Neurons are very difficult cells to transfect. Indeed the most indicated choice would be to infect them with a virus expressing your protein of interest.
If you want to use Lipofectamine you can try, but use Lipofectamine 2000 and not 3000 (it would kill all your cells).
What you need to do is having your neurons in plate (eg. in a 24 well plate on glass coverslips pre-treated with D-poly-Lysine). On transfection day you prepare the transfection mix with: 1ug DNA (concentration used for each 24 wells) + 1.5 uL Lipofectamine 2000 + 200 uL NeuroBasal.
Remove all the medium from the wells and replace with 400uL warm NeuroBasal per well. (NOTE: keep the old medium in a falcon!)
Prepare the mix and vortex everytime you add something. Leave the mix 15 minutes at 37°C.
In the meantime prepare a mix 1:1 of old medium and Neurobasal + 1%gentamicine, filter and keep warm.
Add 200uL per well of transfection mix and leave in incubator for 1h.
After 1 hour remove all the medium and replace with 500ul/well of the 1:1 mix.
Hope this is useful!
You are welcome, Ryan.
Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons.
Calcium phosphate transfection of primary hippocampal neurons - PubMed (nih.gov)
jove-81-50808.pdf (nih.gov)
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