High Ca2+-phosphate transfection efficiency in low-density neuronal cultures

Min Jiang, Gong Chen

Research output: Contribution to journalArticle

192 Citations (Scopus)

Abstract

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).

Original languageEnglish (US)
Pages (from-to)695-700
Number of pages6
JournalNature Protocols
Volume1
Issue number2
DOIs
StatePublished - Jul 1 2006

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Transfection
Toxicity
Phosphates
Neurons
Precipitates
Functional analysis
Particle Size
Culture Media
Buffers
Genes
Particle size
DNA

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. / Jiang, Min; Chen, Gong.

In: Nature Protocols, Vol. 1, No. 2, 01.07.2006, p. 695-700.

Research output: Contribution to journalArticle

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