Neuronal Cell Lines and Primary Neurons Preparation
Primary Neurons Preparation
Primary neurons preparation is a crucial step in order to transfect them efficiently. You will find below the protocol used to prepare hippocampal neurons. In addition, you can find some important tips and tricks in the publication of Buerli, T., Pellegrino, C., Baer, K. et al, “Efficient transfection of DNA or shRNA vectors into neurons using Magnetofection” Nature Protocol 2007 accepted for publication.
A- Solution preparation:
1- Dissection solution: HBSS (without calcium and magnesium) containing 0.25% D-Glucose, keep at 4°C. Prepare fresh solution each time.
2- Culture medium: MEM supplemented with 10% Nu serum, 15mM HEPES pH 7.2, 0.45% glucose, 1mM sodium pyruvate, 2mM L-glutamine, 10 IU/ml penicillin-streptomycin.
3- Culture feeding medium: MEM supplemented with 2% B27, 15mM HEPES, 0.45% glucose, 1mM sodium pyruvate, 2mM glutamine.
B- Tissue culture vessel preparation:
1- Dissolve Poly-L-lysine (Sigma P-1520) at 0.1 mg/mL in water (aliquot and store at -20°C).
2- Cover the coverslip or dish with the Poly-L-lysine solution.
3- Incubate over-night at 37°C.
4- Rinse twice in water.
5- Let dry under a sterile laminar hood.
C- Cell preparation:
Prepared cells from (E)18-19 hippocampal neurons. Use glia-neuron co-culture using defined media and not Banker type cultures nor glia feeder layer. NeuroMag reagent is also suitable with neurobasal media.
1- Rinse hippocampi twice in cooled (0°C, max 2-3°C) dissection solution.
2- Dissect hippocampi free of meninges in cooled (2-3°C) dissection solution.
3- Incubate hippocampi solution with trypsin solution (at 0.25 % final concentration) for 15 min at 37°C in one 15 mL Falcon tube. 10 to 20 hippocampi are generally used in 5 ml of solution in 15 ml falcon tube.
4- Stop the trypsin action by washing two times with HBSS or MEM.
5- Resuspend in a final volume of 1 to 3 mL of MEM containing 10 % fetal calf serum.
6- Triturate tissue using a P1000 micropipette (10 times).
7- Wait 1-2 minutes until non-dissociated tissue goes to the bottom of the tube. Transfer dissociated cells into new tube, add 2 mL of HBSS to the remaining non-dissociated clusters and push it (5-10 times) thought Pasteur pipette until complete dissociation of tissue. Transfer the dissociated cells to the tube containing the first dissociated cells.
8- Count the number of cells.
9- Dilute the dissociated neurons to the desired concentration with culture medium. Plate the cells to the polylysine coated dish or coverslip (see the table 1 below for the suggested cell amount). We recommend optimizing the number of cells relative to the dish/coverslip used. For example, 2mL of cells at a density of 300 000 or 400 000 cells per mL can be plated in 35 mm dish. Cell density will also vary according to the desired time point of transfection (immature or mature).
10- Grow the cells in 5% CO2 and 37°C in the absence of a glial feeder cell layer in 10% Nu tissue culture medium until the desired time point for transfection.
11- From 10 DIV in culture, change 50 % of old medium with fresh culture feeding medium every 3 days and the day before Magnetofection.
Note that optimal neuronal growth prior to Magnetofection is critical. It depends on the addition of B27 in the culture medium and on the cell density.
Neuronal Cell Lines
Neuronal cell lines. It is recommended to seed or plate the cells the day prior transfection. The suitable cell density will depend on the growth rate and the conditions of the cells. Cells should not be less than 60 % confluent (percentage of growth surface covered with cells) at the time of transfection (see the suggested cell number in the table 1). The correct choice of optimal plating density also depends on the planned time between transfection and transgene analysis: for a large interval, we recommend a lower density and for a short interval a higher density may be advantageous. (See section 3.3 for procedure)
Transfection Protocol of DNA or shRNA vectors
The DNA and NeuroMag solutions should have an ambient temperature and be gently vortexed prior to use. The rapid protocol is as simple as follows: Use 3.5 µL of NeuroMag per µg of DNA. We suggest beginning with this ratio and optimize it, if required, by following section 3.6.
1.1. Primary Neurons. Mixed cultured cells were used for this procedure. The cell density is a critical parameter to achieve good transfection with low toxicity; the suitable cell density will depend on the growth rate and the conditions of the cells; higher cell confluency is preferable than low cell density. We recommend optimizing the cell culture density according to your experimental conditions. Primary neurons have been transfected from 7 to 21 DIV. The best results were achieved with cells cultured for 10-15 days in vitro (day of transfection) depending on cell culture conditions.
Exchange 50% of the culture media 24 hours before transfection
1.2. Cell lines. The day prior transfection, prepare the cells as described in section 3.2.
- DNA/NeuroMag complexes preparation
2.1. NeuroMag: Vortex the reagent and place the appropriate amount in a microtube (see Table 2).
2.2. DNA: Dilute the indicated quantity of DNA (see Table 2) in 50 to 700 L of culture medium without serum and supplement.
2.3. Add the DNA solution to the NeuroMag solution by vigorous pipetting or brief vortexing and incubate at room temperature for 15 to 20 minutes.
3.1. Add the NeuroMag / DNA complexes onto cells [growing in culture feeding medium if > 10 DIV or culture medium if <10 DIV] drop by drop and gently rock the plate to ensure a uniform distribution. Place the cell culture plate on the magnetic plate during 15 minutes.
3.2. Remove the magnetic plate.
- Cultivate the cells at 37°C in a CO2 incubator under standard conditions until evaluation of transgene expression (from 24h up to 7 days).
3.4. Optimization Protocol
Although high transfection efficiencies can be achieved with the rapid protocol, some optimization may be needed in order to obtain the maximum efficiency in particular cell lines or primary cells culture. Several parameters can be optimized:
• Dose of nucleic acid used
• Ratio of NeuroMag to nucleic acid
• Cell density
• Incubation time
We recommend that you optimize one parameter at a time. It is required to keep the other parameters (cell number, dose of nucleic acid…) constant while one parameter is being optimized.
- Quantity of DNA:
To achieve the optimal transfection efficiency, the amount of DNA used can be increased or decreased. It is important to always keep the number of cells and the incubation time constant during your optimization procedure. Adjust the best amount of DNA required by maintaining a fixed ratio of NeuroMag reagent to DNA (3.5 µL / µg DNA), and vary the DNA quantity over the suggested range (table 3).
- NeuroMag / DNA ratio:
For optimization, first maintain a fixed quantity of DNA (according to the size of your culture dish, cell number and previous optimization) and then vary the amount of NeuroMag reagent from 2 to 5 µL per 1 µg of DNA.
- Cell number:
The cell number (density) is also a critical parameter to achieve good transfection efficiency and the optimal confluency has to be adjusted according to the cells used. The suitable cell density will depend on the growth rate and the conditions of the cells; higher cell confluency is preferable than low cell density.
- Incubation time:
The optimal time range between transfection and assay for gene activity varies with cells, promoter activity, expression product, etc. The transfection efficiency can be monitored after 24 hours up to several days (7) by analyzing the gene product. Reporter genes such as GFP, β-galactosidase, secreted alkaline phosphatase or luciferase can be used to quantitatively measure gene expression.
In addition, do not hesitate to contact our technical service at [email protected] to request more detailed technical insights and applications update.
3.5. Co-Transfection Protocol
For co-transfection of several plasmids DNA, mix the same amount of each plasmid and transfect as described above in section 3.3 or 3.4. For example, if you have two DNA plasmids, mix 1 µg of each plasmid, complex the 2 µg of DNA with 7 µL of NeuroMag.
3.6. Option for Co-transfection.
Transfections can be realized sequentially instead of simultaneously. So, cells can be transfected with one plasmid DNA first and 4 h to 24 h later can be transfected with the other plasmid DNA. Follow the procedure as detailed above for DNA transfection (3.3 or 3.4). A medium changed can be also performed between the two transfections.