High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.
This protocol describes a high-efficiency Ca2+-phosphate transfection method with low cell toxicity. The Ca2+-phosphate transfection method is widely used in transfecting neurons because of its low cell toxicity and simplicity in use, but the efficiency is typically low (approximately 1-5%). To solve this problem we have developed a new Ca2+-phosphate transfection protocol that increases the efficiency by 10-fold (< or = 60%), while maintaining low cell toxicity. First, it is critical to have gentle mixing of the DNA-Ca2+ solution with phosphate buffer to form a homogeneous snowlike precipitate (particle size 1-3 microm). Second, the precipitate should be dissolved using a slightly acidic culture medium to reduce cell toxicity. The high efficiency of this new protocol makes it possible to transfect single autaptic neurons as well as mature neurons (15-82 days in vitro) for gene functional analysis. The total time required for the protocol is 2-4 h (including 45 min-3 h incubation time).
Jiang
Min
M
Department of Biology, Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Chen
Gong
G
D002130:Calcium Phosphates / Q000378:metabolism*; D002452:Cell Count; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage; D006624:Hippocampus / Q000166:cytology; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D004247:DNA; C020243:calcium phosphate