I was working with lipofectamin 2000 but now had to change to 3000 and the protocol is not working.
Neuronal cell lines should be sufficiently simple to transfect. If you're using an already-established cell line, look for transfection reagents designed for it. Otherwise, use a reagent for neuronal cells (like N2a: https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/) and that should help you increase the overall efficiency.
Dear Tatian,
It is true that a change in reagent can dramatically influence the becoming of your experiment ; and what was true for a version of a reagent may not be true for a new version of a reagent.
That is why I would like to present you a reliable and efficient alternative to classic transfection for primary neurones: NeuroMag
NeuroMag, a Magnetofection reagent (the use of a magnetic field to attract magnetized complexed of nanoparticles/nucleic acid) is a real good alternative.
Numerous paper demonstrated that NeuroMag was highly efficient for transfecting primary neurons with plasmid DNA (even long term cultured neurons) such as:
- hippocampal neurons: Charrier C. et al, Cell 2012 (http://www.ncbi.nlm.nih.gov/pubmed/22559944)
- cortical neurons: Courchet et al, Cell 2013 (http://www.ncbi.nlm.nih.gov/pubmed/23791179)
- or even motor neurons derived ES: Terenzio M et al, EMBO J 2014 (http://www.ncbi.nlm.nih.gov/pubmed/17475624)
Moreover, NeuroMag is also compatible with any nucleic acid (for gene silencing for example) such as:
- siRNA and miRNA in cortical neurons: Absalon S et al, J Neurosc 2013 (http://www.ncbi.nlm.nih.gov/pubmed/24027266)
- shRNA in cortical neurons: Navaladi VC., J Neurosci. 2012 (http://www.ncbi.nlm.nih.gov/pubmed/23100434)
- shRNA in hippocampal neurons: Vargas ML., PLoS One. 2014 (http://www.ncbi.nlm.nih.gov/pubmed/24658113)
would you have any question or would you like to try NeuroMag, please do not hesitate to contact me at: [email protected]
good luck with your experiment,
best regards,
Cedric
May i ask you the reason why you have to change from 2000 to 3000?
How many wells plate are you transfecting? Normally 24 wells plate works the best.
Did you votex the Lipofectamine? Normally I dont vortex it.
What about your incubation time?
Hola Tatiana.
Puesto que eres de Colombia es mejor escribirte directamente en español (disculpando al resto de lectores de este contenido). Resolviste tu problema con la lipo 3000? si es así me podrías comentar cómo...
muchas gracias
Felipe
I find Lipofectamine 3000 better and less toxic than Lipofectamine 2000. Higher efficiency and transfection rate. I use hippocampal neuronal cells. No problem with my experiments.
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