Lipid-Mediated Transfection of Human Astrocytes

Astrocytes are by far the most numerous cell type in the central nervous system (CNS) and have critical roles in adult CNS homeostasis. They provide biochemical and nutritional support of neurons and endothelial cells which form the blood-brain barrier, perform the vast majority of synaptic glutamate uptake, and maintain extracellular potassium levels (Rothstein et al., 1996; Rothstein et al., 1994). Although there are few known differences between cortical and hippocampal astrocytes, it has been reported that astrocytes from different regions of the brain show a differential sensitivity to ischemic injury (Xu et al., 2001; Zhao & Flavin, 2000).

The following protocols provide instructions for lipid-mediated transfection of plasmid DNA or siRNA into Gibco Human Astrocytes using the Lipofectamine LTX Reagent or the Lipofectamine RNAiMAX Reagent. Lipofectamine LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. Lipofectamine RNAiMAX is a proprietary formulation specifically developed for the transfection of siRNA and Stealth RNAi duplexes into eukaryotic cells.

Required materials

• Gibco Human Astrocytes (Cat. No. N7805-100)
• Gibco Astrocyte Medium (Cat. No. A12613-01)
  Note: The medium kit includes N-2 Supplement, 100X (Cat . No. 17502-048), Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (Cat. No. 10569-010), and One Shot Fetal Bovine Serum (FBS), Certified (Cat. No. 16000-077).
• Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X), liquid without Ca²⁺ and Mg²⁺ or phenol red (Cat. No. 14190-144)
• Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X), liquid with Ca²⁺ and Mg²⁺(Cat. No. 14040-133)
• Geltrex Reduced Growth Factor Basement Membrane Matrix (Cat. No. 12760)
• StemPro Accutase Cell Dissociation Reagent (Cat. No. A11105)
• Opti-MEM I Reduced Serum Medium (Cat. No. 31985-062)
• Appropriate tissue culture plates and supplies

For transfecting plasmid DNA

• Plasmid DNA of interest (100 ng/μL or higher)
• Lipofectamine LTX Reagent and PLUS Reagents (Cat. No. 15338-100)

For transfecting siRNA

• Silencer Select siRNAs
• Lipofectamine RNAiMAX Transfection Reagent (Cat. No. 13778-075 or 13778-150)

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Follow these important guidelines when performing lipid-mediated transfections of human astrocytes:

• Maintain human astrocytes on Geltrex matrix–coated plates.
• Adding antibiotics to media during transfection may result in cell death. If you wish to use antibiotics during transfection, test your conditions thoroughly.
• Maintain the same seeding conditions between experiments. Use low-passage cells; make sure that cells are healthy and greater than 90% viable before transfection.
• Transfections can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine LTX or Lipofectamine RNAiMAX Reagent.
• Using PLUS™ Reagent enhances transfection performance in human astrocytes.
• We recommend Opti-MEM I Reduced Serum Medium to dilute the DNA and Lipofectamine LTX Reagent or the siRNA and Lipofectamine RNAiMAX Reagent before complexing.

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Before thawing or passaging Gibco Human Astrocytes, prepare culture vessels coated with Geltrex® matrix as described below.

1. Thaw a bottle of Geltrex Basement Membrane Matrix at 4°C overnight.
   
2. On ice, prepare a stock solution of Geltrex matrix diluted 1:1 in D-MEM. Store in aliquots at –20°C until needed.
   
3. Dilute the stock solution 1:100 in D-MEM and coat the bottom of each culture vessel (200 μL of Geltrex® matrix per cm² of culture vessel).
   
4. Incubate the culture vessel at 37°C for 1 hour. Dishes coated with Geltrex matrix can be used immediately or stored at 4°C for up to a week, sealed with Parafilm. Do not allow dishes to dry. When you are ready to add cells, aspirate the Geltrex matrix solution and rinse the plates once with D-PBS with Ca²⁺ and Mg²⁺ before adding the cell solution.

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Aseptically mix the following components for preparing 100 mL of complete Gibco® Astrocyte Medium. Complete Astrocyte Medium is stable for 2 weeks when stored at 4°C protected from light.

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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1. Warm Complete Astrocyte Medium and StemPro Accutase Cell Dissociation Reagent in a 37°C water bath before use.
   
2. Transfer conditioned medium from the cells to a new tube; this will be used to stop the enzyme reaction in step 6.
   
3. Wash cells once with 1X D-PBS without calcium, magnesium, or phenol red.
   
4. Aspirate D-PBS and add StemPro Accutase to the cells.
   
5. Incubate for 5–10 minutes at 37°C. Rock the cells every ~5 minutes and check under a microscope for detachment and dissociation toward single cells.
   
6. When the cells have detached, add an equal volume (1:1) of conditioned medium (from Step 2) to slow the Accutase activity.
   
7. Transfer the cells to a 15 mL or 50 mL tube.
   
8. Rinse culture vessels with complete medium and add it to the tube.
   
9. Centrifuge the tube for 4 minutes at 200 × g.
   
10. Aspirate and discard the supernatant.
    
11. Gently resuspend the pellet in Complete Astrocyte Medium.
    
12. Count the live cells using a method of choice.
    
13. To replate human astrocytes, remove a Geltrex matrix–coated plate from 4°C storage and tip slightly to aspirate the Geltrex matrix solution. Rinse the plate once with D-PBS with calcium and magnesium. Do not allow the plate to dry out.
    
14. Immediately seed the astrocytes at the desired concentration (we recommend ≥2 × 10⁴ cells/cm²).
    
15. Incubate the cells in an incubator at 37°C in a humidified atmosphere (90%) of 5% CO₂ in air. Change the medium every 2–3 days with fresh Complete Astrocyte Medium.

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Use this procedure to transfect plasmid DNA into Gibco Human Astrocytes using the Lipofectamine LTX Reagent in a 24-well format (for other formats, see Scaling up or down transfections, below). All amounts and volumes are given on a per well basis.

1. The day before transfection, prepare Human Astrocytes that have recovered from cryopreservation and have reached 80% confluency. Use StemPro Accutase to detach the cells and count the cells. Plate 5 × 10⁴ cells per well in 0.5 mL of complete growth medium. Cell density should be 80–90% confluent on the day of transfection.
2. For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μL of Opti-MEM I Reduced Serum Medium without serum.
3. Using PLUS Reagent: Mix PLUS Reagent gently before use, then add 0.5 μL PLUS Reagent (a 1:1 ratio to DNA) directly to the diluted DNA. Mix gently and incubate for 5–15 minutes at room temperature.
4. For each well of cells, dilute 1.5–3.0 μL of Lipofectamine LTX into the above diluted DNA solution, mix gently and incubate for 25 minutes at room temperature to form DNA-Lipofectamine LTX complexes.
5. Remove growth medium from cells and replace with 0.5 mL of complete growth medium. Add 100 μL of the DNA-Lipofectamine LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth.
6. Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO₂ incubator for 18–24 hours post-transfection before assaying for transgene expression.  

Scaling up or down transfections

To transfect human astrocytes in different tissue culture formats, vary the amounts of Lipofectamine LTX Reagent, DNA, cells, medium and PLUS Reagent used in proportion to the relative surface area, as shown in the table (amounts given on a per well basis).

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Prepare siRNAs

1. Resuspend the Silencer Select siRNAs with nuclease-free water. A convenient stock concentration is 100 μM, which can be diluted to meet downstream experimental needs.
2. Validate the concentration of the siRNA by measuring absorption at 260 nM using a spectrophotometer and adjust with water if necessary. Keep aliquots frozen at –20°C.
3. Dilute stock siRNAs of 100 μM to a working concentration of 10 μM.
4. From the working stock dilute siRNAs in 20 μL of Opti-MEM I per well to achieve a final concentration of 30 nM or your desired concentration (1 nM–100 nM) in tubes or plates. When applicable, make master mixes for replicates to minimize variability (at least one well overage). For example with a 10 μM siRNA stock, mix 0.39 μL of siRNA (3.9 pmols) + 19.61 μL of Opti-MEM-I per well or 1.56 μL of siRNA + 78.44 μL of Opti-MEM-I for the master mix.
5. Plate siRNAs in a Geltrex matrix–coated plate using the proper method for coating as provided by the manufacturer
   

Prepare cells

Culture cells according to manufacturer's cell protocol. Cells were in culture for about a week. On the day of transfection, harvest cells according to the protocol. Count and dilute cells to the proper density. We recommend performing an initial optimization experiment to determine your optimal cell density. Based on our optimization, we found 4,000 cells per well to be an optimal cell density.

Transfect cells

We recommend performing an initial optimization experiment to determine the optimal amount of transfection agent to add that balances good knockdown and low toxicity. Based on our optimizations, we found 0.15 μL per well of Lipofectamine RNAiMAX to be the best condition.

1. Dilute 0.15 μL per well of Lipofectamine RNAiMAX in Opti-MEM I for a total volume of 10 μL per well in a polystyrene 12 × 75-mm tube or a conical centrifuge tube. Make a master mix of sufficient volume to treat all wells to be transfected plus an extra 10% for pipetting variability. Mix the Lipofectamine RNAiMAX–Opti-MEM I mixture by gently flicking the bottom of the tube.
2. Combine 10 μL of the Lipofectamine RNAiMAX–Opti-MEM I mixture per 20 μL of diluted and pre-plated siRNA. Mix by tapping the plate back and forth. Incubate this mixture for 10 minutes at room temperature.
3. After the incubation, add 80 μL of human astrocytes that have been diluted to the proper density to each well. The final volume per well should be 110 μL per well.
4. Place the plate in a 37°C incubator under normal cell culture conditions. Remove the cells and assay for the expression levels of the gene of interest at the desired time point (typically 24–48 hours post-transfection).

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1. Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol, et al. (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16:675–686.
   
2. Rothstein JD, Martin L, Levey AI, et al. (1994) Localization of neuronal and glial glutamate transporters. Neuron 13:713–725.
   
3. Xu L, Sapolsky RM, Giffard RG (2001) Differential sensitivity of murine astrocytes and neurons from different brain regions to injury. Exp Neuro 169:416–424.
   
4. Zhao G, and Flavin MP (2000) Differential sensitivity of rat hippocampal and cortical astrocytes to oxygen-glucose deprivation injury. Neurosci Lett 285:177–180.