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 (∼1-5%). To solve this problem we have developed a new Ca²⁺-phosphate transfection protocol that increases the efficiency by 10-fold (≤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 μm). 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).