title: pH dependent high transfection efficiency of mouse neuroblastomas using TransFectin.
abstract: Mouse neuroblastoma cell lines are often used in lieu of mouse primary neurons in ex vivo experiments, as they provide an easier platform for transfection, compared to the latter. A well-known inherent problem with this strategy is the relatively low transfection efficiency (15-30%) of mouse neuroblastoma cell lines such as neuro-2A and N1E-115. We were able to improve the transfection efficiency of these cell lines by using the cationic lipid reagent, TransFectin (Bio-Rad, Hercules, CA, USA) to optimise the transfection conditions. Our results, based on fluorescence intensity determinations and Western blotting for enhanced green fluorescence protein (EGFP) over-expression in neuro-2A, demonstrated that pH is a crucial factor in determining the transfection efficiency. Under pH-optimised transfection conditions, flow cytometric analysis revealed high EGFP transfection efficiencies of 76.4 +/- 0.5 and 60.9 +/- 0.6% for neuro-2A and N1E-115, respectively. Notably, the optimised TransFectin-based transfection system did not result in any detectable cytotoxicity to the mouse neuroblastomas. The resultant optimised system is economical, easy to use and does not require any specialised equipment.
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore.
authors.forename: Kevin Wai Yin
authors.initials: KW
authors.lastname: Chong
authors.forename: Alan Yiu-Wah
authors.initials: AY
authors.lastname: Lee
authors.forename: Evelyn S C
authors.initials: ES
authors.lastname: Koay
authors.forename: Sze Jee
authors.initials: SJ
authors.lastname: Seet
authors.forename: Nam Sang
authors.initials: NS
authors.lastname: Cheung
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002412:Cations / Q000494:pharmacology*; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000494:pharmacology*; D051379:Mice; D009447:Neuroblastoma; D013997:Time Factors; D014162:Transfection / Q000379:methods*
chemical_list: D002412:Cations; D008081:Liposomes; D049452:Green Fluorescent Proteins