Transfecting Neural Cells Using the Neon® Transfection System

Transfecting Neural Cells Using the Neon Transfection System<A name="top"></A>

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Introduction
Culture Conditions
Preparing Media
Transfection Protocol
Expected Results
Troubleshooting

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 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.

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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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 Cells	Complete 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 Astrocytes	Complete 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 Cells	Complete 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 Cells	Complete 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-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

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-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.

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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.

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.
On the day of the experiment, harvest and wash cells in phosphate buffered saline (PBS) without Ca²⁺ and Mg²⁺.
Resuspend the cell pellet in Resuspension Buffer R (included with Neon Kits) at the appropriate final density (see the following table).
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.

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 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

Fill the Neon Tube with 3 mL of Buffer E. (Use Buffer E2 if you are using the 100-μL Neon Tip.)
Insert the Neon Tube into the Neon Pipette Station until you hear a click, indicating that the tube has locked in position.
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.
Add 1 mL of cells (resuspended in step 3) to the tube containing the plasmid DNA and gently mix.
Insert a 10-μL Neon Tip into the Neon Pipette.
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.
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.
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.
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.
Repeat Steps 10–14 for the remaining samples.
Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37°C in a humidified 5% CO₂ incubator.
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).

A	B

Cell density	Pulse voltage (V)	Pulse width (ms)	Pulse number	Transfection efficiency	Viability	Neon tip
1 × 10⁷ cells/mL	1400 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).

A	B

Cell density	Pulse voltage (V)	Pulse width (ms)	Pulse number	Transfection efficiency	Viability	Neon tip
1 × 10⁷ cells/mL	1100 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).

A	B

Cell density	Pulse voltage (V)	Pulse width (ms)	Pulse number	Transfection efficiency	Viability	Neon tip
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

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).

A	B

Cell density	Pulse voltage (V)	Pulse width (ms)	Pulse number	Transfection efficiency	Viability	Neon tip
1 × 10⁷ cells/mL	1300 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 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	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 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	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

Transfecting Neural Cells Using the Neon Transfection System