High Ca2+-phosphate Transfection Efficiency in Low-Density Neuronal Cultures

Nat Protoc. 2006;1(2):695-700. doi: 10.1038/nprot.2006.86.

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 (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).

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calcium Phosphates / metabolism*
  • Cell Count
  • Cells, Cultured
  • DNA / administration & dosage
  • Hippocampus / cytology
  • Neuroglia / metabolism
  • Neurons / cytology*
  • Neurons / metabolism*
  • Transfection / methods*

Substances

  • Calcium Phosphates
  • DNA
  • calcium phosphate