Protocol

Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid

This protocol was adapted from "Imaging the Actin Cytoskeleton," Chapter 29, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. One to two percent transfected cells is a typical result. A disadvantage of the calcium phosphate method is that hippocampal neurons become "fragile" after treatment.