Introduction

The Neon® Transfection System is a benchtop electroporation device that uses the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary cells and stem cells. Instructions for using the Neon® Transfection System for transfecting of neural cells are described below. For detailed instructions on using the Neon® Transfection System, refer to the manual supplied with the product or download the manual from www.invitrogen.com. For detailed information on culture conditions for various neural cell lines, refer to the instructions supplied with the specific cell line you are using.


Required Materials

  • Neural cell line of interest
  • Growth media and growth factors appropriate for your neural cell line
  • Plasmid DNA of interest (1–5 μg/mL in deionized water or TE)
  • Dulbecco’s Phosphate-Buffered Saline (D-PBS) (1X), liquid without Ca2+ and Mg2+ (Cat. no. 14190-144)
  • Neon® Transfection system (Cat. no. MPK5000)
  • Neon® Kit, 10 μL (Cat. no. MPK1096) or Neon® Kit, 100 μL (Cat. no. MPK10096)
  • Appropriate tissue culture plates and supplies
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Ordering Information

Sku Name Size Price Qty
A1261301 Gibco® Astrocyte Medium 500 mL USD 112.00
N7805100 Gibco® Human Astrocytes 1 mL USD 712.00
MPK5000 Neon® Transfection System 1 each USD 5,090.00
MPK1096 Neon® Transfection System 10 µL Kit 96 × 2 reactions USD 1,404.00
MPK10096 Neon™ Transfection System 100 µL Kit 96 × 2 reactions USD 1,404.00

Culture Conditions

The following table summarizes the culture conditions for various neural cell lines, including neural stem cells. For detailed instructions on culturing and passaging these cells, refer to the to the instructions supplied with the specific cell line you are using.

Cell type Media  Culture conditions
Human Neural Stem CellsComplete StemPro® NSC SFM
  • Adherent culture on CELLStart™-, fibronectin-, or poly-L-ornithine-coated culture vessels
  • 37°C, humidified atmosphere of 5% CO2 in air
  • Exchange spent medium every other day
Human AstrocytesComplete GIBCO® Astrocyte Medium
  • Adherent culture on Geltrex™-coated tissue culture vessels
  • 37°C, humidified atmosphere of 5% CO2 in air
  • Exchange spent medium every 3–4 days
Rat Fetal Neural Stem CellsComplete StemPro® NSC SFM
  • Adherent culture on CELLStart™-, fibronectin-, or poly-L-ornithine-coated culture vessels
  • 37°C, humidified atmosphere of 5% CO2 in air
  • Exchange spent medium every 3–4 days
Rat Primary Cortical Astrocytes
Complete GIBCO® Astrocyte Medium*
  • Adherent culture on standard culture vessels
  • 37°C, humidified atmosphere of 5% CO2 in air
  • Exchange spent medium every 2–3 days
Rat Glial Precursor CellsComplete StemPro® NSC SFM,
supplemented with 10 ng/mL PDGF-AA
  • Adherent culture on CELLStart™- or poly-L-ornithine-coated culture vessels
  • 37°C, humidified atmosphere of 5% CO2 in air
  • Exchange spent medium every other day

*For increased proliferation of rat astrocytes, you can supplement complete GIBCO® Astrocyte Medium (D-MEM with 1X N-2 Supplement and 10% OneShot™ FBS) with 20 ng/mL EGF. Adding EGF to human astrocyte cultures can increase proliferation, but may result in morphological or phenotypic changes.
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Preparing Media

Complete StemPro® NSC SFM

To prepare 100 mL of complete StemPro® NSC SFM, aseptically mix the components listed in the table below. Complete medium is stable for up to 4 weeks when stored in the dark at 4°C.

Component Concentration Amount
KnockOut™ D-MEM/F-121X97 mL
GlutaMAX™-I Supplement2 mM1 mL
bFGF20 ng/mL2 μg
EGF20 ng/mL2 μg
NSC SFM Supplement2%2 mL



Complete GIBCO® Astrocyte Medium


To prepare 100 mL of complete GIBCO® NSC SFM, aseptically mix the components listed in the table below. Complete medium is stable for up to 2 weeks when stored in the dark at 4°C

Component Concentration Amount
D-MEM1X89 mL
N-2 Supplement1X1 mL
FBS10%10 mL


Note: Adding EGF at a final concentration of 20 ng/mL can increase proliferation, but may result in morphological and phenotypic changes in human astrocytes.

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Transfection Protocol

Use this procedure to transfect plasmid DNA into hNSCs in a 24-well format using the 10-μL Neon® Kit. All amounts and volumes are given on a per well basis.

  1. Cultivate the required number of cells in the appropriate growth medium (see table below) such that the cells are 70–90% confluent on the day of the experiment.
  2. On the day of the experiment, harvest and wash cells in phosphate buffered saline (PBS) without Ca2+ and Mg2+.
  3. Resuspend the cell pellet in Resuspension Buffer R (included with Neon® Kits) at the appropriate final density (see the following table).
  4. Prepare 24-well plates by filling the wells with 0.5 mL of the appropriate growth medium without antibiotics and pre-incubate plates at 37°C in a humidified 5% CO2 incubator. If using other plate formats, adjust the volume accordingly.
  5. Turn on the Neon® unit and enter the following electroporation parameters in the Input window. Alternatively, press the Database button and select the appropriate transfection protocol (if you have already added the electroporation parameters for your cell type). For detailed instructions, refer to the manual supplied with the Neon® unit.

    Cell type Cell density Pulse voltage (V) Pulse width (ms) Pulse number  
    Neon® tip
    Human Neural Stem Cells1 × 107 cells/mL1400
    1600
    1700
    20
    20
    20
    2
    1
    1
    10-μL
    Human Astrocytes1 × 107 cells/mL1100
    1200
    30
    40
    1
    1
    10-μL
    Rat Fetal Neural Stem Cells1 × 107 cells/mL1300
    1500
    1600
    20
    10
    10
    2
    3
    3
    10-μL
    Rat Primary Cortical Astrocytes0.5 × 107 cells/mL1400
    1400
    1700
    20
    30
    20
    2
    1
    1
    10-μL

    Rat Glial Precursor Cells

     1 × 107 cells/mL

    1300
    1500

    10
    20

    3
    1
     
    10-μL

  6. Fill the Neon® Tube with 3 mL of Buffer E. (Use Buffer E2 if you are using the 100-μL Neon® Tip.)
  7. Insert the Neon® Tube into the Neon® Pipette Station until you hear a click, indicating that the tube has locked in position.
  8. Transfer 0.5 μg of plasmid DNA into a sterile, 1.5-mL microcentrifuge tube. Note: The quality and concentration of DNA used for electroporation plays an important role for the transfection efficiency. We strongly recommend using high quality plasmid purification kits such as PureLink™ HiPure Plasmid DNA Purification Kits to prepare DNA.
  9. Add 1 mL of cells (resuspended in step 3) to the tube containing the plasmid DNA and gently mix.
  10. Insert a 10-μL Neon® Tip into the Neon® Pipette.
  11. Press the push-button on the Neon® Pipette to the first stop and immerse the Neon® Tip into the cell-DNA mixture. Slowly release the push-button on the pipette to aspirate the cell-DNA mixture into the Neon® Tip.
  12. Insert the Neon® Pipette with the sample vertically into the Neon® Tube placed in the Neon® Pipette Station until you hear a click, indicating that the pipette has locked in position.
  13. Ensure that you have entered the appropriate electroporation parameters and press Start on the Neon® touchscreen. The Neon® device delivers the electric pulse according to the parameters entered in step 5 and the touchscreen displays Complete to indicate that electroporation is complete.
  14. Remove the Neon® Pipette from the Neon® Pipette Station and immediately transfer the samples from the Neon® Tip into the prepared culture plate containing the appropriate pre-warmed complete growth medium without antibiotics. Discard the Neon® Tip into an appropriate biological hazardous waste container.
  15. Repeat Steps 10–14 for the remaining samples.
  16. Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37°C in a humidified 5% CO2 incubator.
  17. Assay the samples to determine the transfection efficiency (e.g., fluorescence microscopy or functional assay).
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Expected Results

Human Neural Stem Cells

GIBCO® Human Neural Stem Cells (Cat. no. N7800-100), cultured in StemPro® NSC SFM complete medium, were transfected with 0.5 μg of a plasmid encoding the Emerald Green Fluorescent Protein (EGFP) using the Neon® Transfection system with the parameters listed in the following table. 48 hours post-transfection, the cells were analyzed by light (Panel A) and fluorescence microscopy (Panel B).

AB

 

Cell density Pulse voltage (V) Pulse width (ms) Pulse number Transfection efficiency Viability Neon® tip
1 × 107 cells/mL1400
1600
1700
20
20
20
2
1
1
82%
84%
87%
95%
95%
96%
10-μL



Human Astrocytes

GIBCO® Human Astrocytes (Cat. no. N7805-100) were transfected using the Neon® Transfection Device and 0.5 μg of a plasmid encoding the Emerald Green Fluorescent Protein (EGFP); 24 hours post-electroporation, the cells were analyzed by light (Panel A) and fluorescence microscopy (Panel B).

AB

 

Cell density Pulse voltage (V) Pulse width (ms) Pulse number Transfection efficiency Viability Neon® tip
1 × 107 cells/mL1100
1200
30
40
1
1
92%
93%
97%
97%
10-μL



Rat Fetal Neural Stem Cells

GIBCO® Rat Fetal Neural Stem Cells (Cat. no. N7744-100) were transfected using the Neon® Transfection Device and 0.5 μg of a plasmid encoding the Emerald Green Fluorescent Protein (EGFP); 24 hours post-electroporation, the cells were analyzed by light (Panel A) and fluorescence microscopy (Panel B).

AB

 

Cell density Pulse voltage (V) Pulse width (ms) Pulse number Transfection efficiency Viability Neon® tip
1 × 107 cells/mL1100
1200
30
40
1
1
92%
93%
97%
97%
10-μL



Rat Primary Cortical Astrocytes

GIBCO® Rat Primary Cortical Astrocytes (Cat. no. N7745-100) were transfected using the Neon® Transfection Device and 0.5 μg of a plasmid encoding the Emerald Green Fluorescent Protein (EGFP); 24 hours electroporation, the cells were analyzed by light (Panel A) and fluorescence microscopy (Panel B).

AB

 

Cell density Pulse voltage (V) Pulse width (ms) Pulse number Transfection efficiency Viability Neon® tip
0.5 × 107 cells/mL1400
1400
1700
20
30
20
2
1
1
69%
71%
71%
87%
89%
90%
10-μL



Rat Glial Precursor Cells

GIBCO® Rat Glial Precursor Cells (Cat. no. N7746-100) were transfected using the Neon® Transfection Device and 0.5 μg of a plasmid encoding the Emerald Green Fluorescent Protein (EGFP); 24 hours post-electroporation, the cells were analyzed by light (Panel A) and fluorescence microscopy (Panel B).

AB

 

Cell density Pulse voltage (V) Pulse width (ms) Pulse number Transfection efficiency Viability Neon® tip
1 × 107 cells/mL1300
1500
10
20
3
1
49%
44%
78%
64%
10-μL

 

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Troubleshooting

For troubleshooting tips regarding the culture and passaging of your cells, refer to the manual provided with the cells. For troubleshooting tips regarding the Neon® Transfection System, see below.

Problem Possible cause
Solution
Connection failureNo Neon® Tip is inserted or
the Neon® Tip is inserted
incorrectly
Make sure that the Neon® Tip is inserted into Neon® Pipette correctly as described. There should be no gap between the tip and the top head of the pipette
Arcing (sparks)Air bubbles in the Neon® Tip
Avoid any air bubbles in the Neon® Tip while aspirating the sample.

High voltage or pulse length
settings
Reduce the voltage or pulse length settings.
Low cell survival ratePoor DNA quality

  • Use high quality plasmid DNA for transfection (use high quality plasmid purification kits such as PureLink™ HiPure Plasmid DNA Purification Kits, Cat. no. K2100) to prepare DNA.
  • Resuspend the purified DNA in deionized water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at a concentration between 0.5–5 μg/μL.
  • Check the purity of the purified DNA preparation by measurement of the A260/280 ratio. The ratio should be at least 1.8 for electroporation.
  • Do not precipitate DNA with ethanol to concentrate DNA. Concentrated DNA by ethanol precipitation shows poor transfection efficiency and cell viability due to salt contamination.

Cells are stressed or damaged
  • Avoid severe conditions during cell harvesting especially high speed centrifugation and pipette cells gently.
  • Avoid using over confluent cells or cells at high densities as this may affect the cell survival after electroporation.
  • After electroporation, immediately plate the cells into prewarm culture medium without antibiotics

Multiple use of the same Neon® TipDo not use the same Neon® Tip for electroporation for more than 2 times because the repeated application of electric pulses reduce the tip quality and impair their physical integrity.
Low transfection
efficiency
Poor plasmid DNA quality or the plasmid DNA is low
  • Use high quality plasmid DNA for transfection.
  • Start with 0.5 μg plasmid DNA per sample.

Incorrect cell density
Use the recommended cell densities of 1 × 105 cells per 10 μL per sample (i.e., 1 × 107 cells/mL).

Incorrect electroporation
parameters
Use the recommended voltage, pulse width, and pulse number. We recommend optimizing the electroporation parameters using the preprogrammed 24-well optimization protocol available on the Neon® unit.

Mycoplasma contaminated
cells
Test cells for Mycoplasma contamination. Start a new culture from a fresh stock
Nonreproducible
transfection
efficiency
Inconsistent cell confluency
or passage number
Always use cells with low passage number and harvest cells with comparable confluency levels.

Multiple use of the same Neon® Tip or the same Neon® Tube
  • Do not use the same Neon® Tip for more than 2 times because the repeated application of electric pulses reduce the tip quality and impair their physical integrity.
  • Do not use the same Neon® Tube for more than 10 times.
  • Always use a new Neon® Tip and Neon® Tube for different plasmid DNA samples to avoid any cross-contamination.
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LT163                    17-Mar-2011