海马神经元细胞转染的实验技术方法.pdfVIP

中国试剂网 1 海马神经元细胞转染的实验技术方法 ABSTRACT 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. MATERIALS Reagents Actin-GFP plasmid DNA (purified) BES-buffered saline (BBS), 2X (for calcium phosphate method) BES, 50 mM Na2HPO4, 1.5 mM NaCl2, 280 mM CaCl2, 250 mM (for calcium phosphate method) DOTAP (Roche Diagnostics) Glial monolayer culture (Spector et al. 1998a) Glial-conditioned medium Glucose Hanks balanced salt solution (HBSS); Ca++-Mg++-free (also available from GIBCO) (for DOTAP method) NaCl, 8.0g/liter KCl, 0.4g/liter KH2PO4, 60 mg/liter Na2HPO4 anhydrous, 47.86 mg/liter glucose anhydrous, 1g/liter NaHCO3, 0.35g/liter 中国试剂网 1 Sterilize and refrigerate. Modified HEPES-buffered saline (MHBS), prewarmed (for calcium phosphate method) 135 mM NaCl 1 mM Na2HPO4 4 mM KCl 2 mM CaCl2 20 mM HEPES buffer (Sigma) Make up 100 ml, adjust the pH to 7.5, and filter-sterilize. Horse serum (for DOTAP method) Modified Eagles medium (MEM), (Invitrogen) (for DOTAP method) Neurons (freshly prepared from E18 rat hippocampus for DOTAP method, or growing on coverslips for calcium phosphate method) Equipment Bacterial culture dish, 10-cm diameter (for DOTAP method) Conical tube, 15-ml polystyrene (for DOTAP met