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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 product manual. For detailed information on culture conditions for various neural cell lines, refer to the instructions supplied with the specific cell line you are using.
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 |
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Human Neural Stem Cells | Complete StemPro NSC SFM |
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Human Astrocytes | Complete GIBCO Astrocyte Medium |
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Rat Fetal Neural Stem Cells | Complete StemPro NSC SFM |
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Rat Primary Cortical Astrocytes | Complete GIBCO Astrocyte Medium* |
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Rat Glial Precursor Cells | Complete StemPro NSC SFM, supplemented with 10 ng/mL PDGF-AA |
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*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. |
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 |
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KnockOut D-MEM/F-12 | 1X | 97 mL |
GlutaMAX-I Supplement | 2 mM | 1 mL |
bFGF | 20 ng/mL | 2 μg |
EGF | 20 ng/mL | 2 μg |
NSC SFM Supplement | 2% | 2 mL |
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-MEM | 1X | 89 mL |
N-2 Supplement | 1X | 1 mL |
FBS | 10% | 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.
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.
Cell type | Cell density | Pulse voltage (V) | Pulse width (ms) | Pulse number | Neon tip |
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Human Neural Stem Cells | 1 × 107 cells/mL | 1400 1600 1700 | 20 20 20 | 2 1 1 | 10-μL |
Human Astrocytes | 1 × 107 cells/mL | 1100 1200 | 30 40 | 1 1 | 10-μL |
Rat Fetal Neural Stem Cells | 1 × 107 cells/mL | 1300 1500 1600 | 20 10 10 | 2 3 3 | 10-μL |
Rat Primary Cortical Astrocytes | 0.5 × 107 cells/mL | 1400 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 |
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).
A | B |
Cell density | Pulse voltage (V) | Pulse width (ms) | Pulse number | Transfection efficiency | Viability | Neon tip |
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1 × 107 cells/mL | 1400 1600 1700 | 20 20 20 | 2 1 1 | 82% 84% 87% | 95% 95% 96% | 10-μL |
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).
A | B |
Cell density | Pulse voltage (V) | Pulse width (ms) | Pulse number | Transfection efficiency | Viability | Neon tip |
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1 × 107 cells/mL | 1100 1200 | 30 40 | 1 1 | 92% 93% | 97% 97% | 10-μL |
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).
A | B |
Cell density | Pulse voltage (V) | Pulse width (ms) | Pulse number | Transfection efficiency | Viability | Neon tip |
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1 × 107 cells/mL | 1100 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).
A | B |
Cell density | Pulse voltage (V) | Pulse width (ms) | Pulse number | Transfection efficiency | Viability | Neon tip |
---|---|---|---|---|---|---|
0.5 × 107 cells/mL | 1400 1400 1700 | 20 30 20 | 2 1 1 | 69% 71% 71% | 87% 89% 90% | 10-μL |
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).
A | B |
Cell density | Pulse voltage (V) | Pulse width (ms) | Pulse number | Transfection efficiency | Viability | Neon tip |
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1 × 107 cells/mL | 1300 1500 | 10 20 | 3 1 | 49% 44% | 78% 64% | 10-μL |
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 |
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Connection failure | No 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 rate | Poor DNA quality |
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Cells are stressed or damaged |
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Multiple use of the same Neon Tip | Do 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 |
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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 |
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