Transfection of Cultured Primary Neurons via Nucleofection

Manuel Zeitelhofer1, John P. Vessey1, Sabine Thomas1, Michael Kiebler1, Ralf Dahm1

1 Medical University of Vienna, Center for Brain Research, Vienna, Austria
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.32
DOI:  10.1002/0471142301.ns0432s47
Online Posting Date:  April, 2009
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Abstract

Despite the development of various transfection methods, the transfection of post‐mitotic cells, including neurons, poses a challenging task. Nucleofection, a specialized form of electroporation described in this unit, achieves high transfection efficiencies in primary mammalian neurons, such as hippocampal neurons, while simultaneously maintaining high cell viability. Therefore, it allows for biochemical analyses that rely on large numbers of transfected cells. The recently developed 96‐well shuttle system described in this unit further permits the transfection of up to 96 different constructs in a single experiment. This opens up the possibility for large‐scale experiments in primary neurons, such as shRNA‐mediated knock‐down of a wide range of target genes. Curr. Protoc. Neurosci. 47:4.32.1‐4.32.21. © 2009 by John Wiley & Sons, Inc.

Keywords: transfection; nucleofection; primary hippocampal neurons; transfection efficiency; expression plasmids; RNAi knock‐down

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transfection by Nucleofection
  • Alternate Protocol 1: Nucleofection of Primary Hippocampal Neurons with the 96‐Well Shuttle
  • Support Protocol 1: Preparation of Glial Support Cultures
  • Support Protocol 2: Preparation of Poly‐L‐Lysine‐Coated Coverslips and Dishes
  • Support Protocol 3: Preparation of Dissociated Hippocampal Neurons from Embryonic Brains
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Transfection by Nucleofection

  Materials
  • B27‐supplemented neuronal MEM (NMEM‐B27; see recipe)
  • Rat neuron nucleofector kit, mouse neuron nucleofector kit or small‐cell‐number basic neuron nucleofector kit (kits available from Amaxa) containing:
    • Cuvettes
    • Plastic pipets
    • pmaxGFP control expression plasmid
    • Nucleofector solution, store at 4°C
    • Nucleofector supplement, store at 4°C
  • TE buffer (see recipe)
  • Freshly isolated neurons (see protocol 5)
  • DMEM‐HS (see recipe), 37°C
  • 6‐cm cell culture–grade plastic dishes coated with poly‐L‐lysine (see protocol 4)
  • Glass coverslips, 15‐mm diameter, coated with poly‐L‐lysine (see protocol 4), optional
  • 36.5°C, 5% CO 2 humidified tissue culture incubator
  • Sterile 1.5‐ml microcentrifuge tubes
  • Nucleofector device (Nucleofector IIS, Amaxa)

Alternate Protocol 1: Nucleofection of Primary Hippocampal Neurons with the 96‐Well Shuttle

  Materials
  • NMEM‐B27 or DMEM (see reciperecipes)
  • Rat neuron 96‐well nucleofector kit (Amaxa) containing:
    • 2 × 8 nucleofection modules
    • pmaxGFP control expression plasmid
    • Nucleofector solution, store at 4°C
    • Nucleofector supplement (only needed if using the 96‐well shuttle), store at 4°C
  • TE buffer (see recipe)
  • E15.5 mouse primary hippocampal neurons
  • Sterile conventional 96‐well plates coated with poly‐L‐lysine (see protocol 4)
  • 36.5°C, 5% CO 2 incubator
  • Multi‐channel pipettor with 8 channels and 10‐ to 100‐µl capacity
  • epT.I.P.S. pipet tips (Eppendorf) for multi‐channel pipettor
  • 96‐well shuttle nucleofector (Amaxa)
  • Poly‐L‐lysine‐coated glass coverslips in equilibrated (36.5°C, 5% CO 2), B27‐supplemented NMEM‐containing 12‐well plates (see protocol 4), optional

Support Protocol 1: Preparation of Glial Support Cultures

  Materials
  • Hanks' balanced salt solution (HBSS; see recipe), 37°C
  • Trypsin‐EDTA solution (see recipe)
  • Pregnant rats or mice carrying E17 and E15.5 embryos, respectively
  • Non‐toxic disinfectant with a broad spectrum of bactericidal, fungicidal, and anti‐viral activity, e.g., Barrycidal 36 (Biohit Deutschland)
  • 70% ethanol
  • DMEM‐HS (see recipe)
  • Phosphate‐buffered saline (PBS, see recipe), 37°C
  • B27‐supplemented neuronal MEM (NMEM‐B27; see recipe)
  • Fetal bovine serum (FBS)
  • 37°C water bath
  • Laminar flow hood (class II preferred)
  • Large scissors
  • Straight and large forceps
  • Sterile 3‐, 6‐, and 10‐cm tissue culture dishes
  • Fine scissors
  • Spatula, sterile
  • Vannas spring scissors
  • Sterile 15‐ml conical tubes
  • Sterile 10‐ml pipets
  • Cell culture flasks
  • 36.5°C, 5% CO 2 tissue culture incubator
  • Stereomicroscope with bright‐field‐transmission illumination achieving magnifications of up to 200× (combined ocular‐objective magnification)
  • Hemacytometer
  • Centrifuge with rotor accommodating 15‐ml conical tubes
  • Cryovials

Support Protocol 2: Preparation of Poly‐L‐Lysine‐Coated Coverslips and Dishes

  Materials
  • 65% nitric acid
  • Sterile, distilled water
  • Paraffin pellets
  • Poly‐L‐lysine hydrobromide powder
  • Borate buffer
  • DMEM‐HS (see recipe)
  • B27‐supplemented neuronal MEM (NMEM‐B27; see recipe)
  • Glass coverslips or culture dishes or multi‐well plates
  • Porcelain staining racks for coverslips
  • Glass container
  • 70°C oven
  • Dry‐heat oven for sterilization
  • Sterile 6‐cm cell culture–grade plastic dishes
  • 50‐ml glass bottles
  • Heating plate
  • Sterile glass Pasteur pipets
  • 0.22‐µm pore size filters
  • 36.5°C, 5% CO 2 humidified incubator

Support Protocol 3: Preparation of Dissociated Hippocampal Neurons from Embryonic Brains

  Materials
  • Brain hemispheres (see protocol 3, steps 1 to 9)
  • Hanks' balanced salt solution (HBSS; see recipe), 37°C
  • Trypsin‐EDTA solution (see recipe), 37°C
  • DMEM‐HS (see recipe), 37°C
  • Sterile 6‐cm cell culture‐grade plastic dish
  • Stereomicroscope with brightfield‐transmission illumination achieving magnifications of up to 200× (combined ocular‐objective magnification)
  • Vannas spring scissors
  • Straight forceps
  • Sterile 15‐ml conical tubes
  • 37°C water bath
  • Sterile Pasteur pipets (normal and fire‐polished)
  • Hemacytometer
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Figures

Videos

Literature Cited

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   Hoshimaru, M., Ray, J., Sah, D.W., and Gage, F.H. 1996. Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v‐myc oncogene. Proc. Natl. Acad. Sci. U.S.A. 93:1518‐1523.
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   Vessey, J.P., Vaccani, A., Xie, Y., Dahm, R., Karra, D., Kiebler, M.A., and Macchi, P. 2006. Dendritic localization of the translational repressor Pumilio 2 and its contribution to dendritic stress granules. J. Neurosci. 26:6496‐6508.
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   Zeitelhofer, M., Karra, D., Vessey, J.P., Jaskic, E., Macchi, P., Thomas, S., Riefler, J., Kiebler, M., and Dahm, R. 2009. Improved protocol for high‐efficiency transfection of shRNA‐encoding plasmids into primary hippocampal neurons. J. Neurosci. Res. 87:289–300.
Internet Resources
  http://www.amaxa.com/cell‐database.html
  For information on protocols for the nucleofection of various cell types, refer to the collection of protocols provided on the manufacturer's Web page.
  http://www.amaxa.com/citations.html
  The collection of publications describing the use of the nucleofection technology on the manufacturer's Web page.
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