Nucleofection of Primary Fibroblast Cells
(Transfection using NucleofectorTM)
Materials
  1. Cells to be transfected (NDF: Normal dermal fibroblast)
  2. Basic Nucleofector Kit - Primary Fibroblasts, Cat# D-VPI-1002 which contains:
    • 450ul Nucleofector(TM) Solution
    • 100 ul Supplement
    • 5 certified cuvettes
    • 5 plastic pipettes
    • 20 ul pmaxGFP @ 0.5ug/ul
    • Stored at 4oC

    or

    VPI-1002 which contains:

    • 2.25 ml Nucleofector(TM) Solution
    • 0.5 ml Supplement
    • 25 certified cuvettes
    • 25 plastic pipettes
    • Stored at 4oC
  3. Control vector
  4. Plasmid DNA need to be transfected (purified with Qiagen endotoxic free (concentration should be higher than 1ug/ul)
  5. Growth media (DMEM + 10% FBS + 1% 100X PenStrep)
  6. Trypsin-EDTA solution
  7. Sterile PBS
  8. Amaxa Biosystems Nucleofector II, AAD-1001N, Device No.300 140 (Program U-23)
  9. 50ml tubes
  10. 1.5 ml Eppendorf tubes
  11. Tissue culture flasks or dishes (For stable transfection, dishes are used so you can pick colonies later)
  12. Pippetters
  13. Hemacytometer
  14. phase microscope
  15. Centrifuge (capible of holding 50 ml tubes and spinning at 200xg)
  16. Incubator for cell culture
  17. Hood
Procedure
  1. Grow cells 2-3 days before nucleofection. On the day of nucleofection, the cells should reach to 80-90% confluency.
      2. For each nucleofection, 0.5-2.0 x 106 cells/reaction should be used and using positive (pmaxGFP) and negtive (without DNA) controls is recommended.
  2. Pre-warm Nucleofection Solutions to room temperature.
  3. Prepare nucleofector work solution by adding the appropriate Supplement into corresponding Nucleofector solution at the ratio of 1:4.5 and mix gently (a very brief vortexing won't hurt).
  4. For each sample, put 500ul medium into an sterile Eppendorf tube and pre-warm at 37oC in incubator.
  5. Add 8ml growth medium into a 10cm tissue culture dish for each sample and pre-warm at 37oC in incubator.
  6. Add 2-5ug each plasmid DNA in 1.5 ml sterile Eppendorf tubes individually.
  7. Trypsinize the cells with trypsin-EDTA at 3ml/10cm dish or 3.5ml/T75 flask, DO NOT over trypsinized. Add 7ml growth medium containing 10% FBS to neutralise the trypsin. Pippet up and down couple of times to break cell clumps.
  8. Count cell number using hemacytometer under microscope and calculate cell density in the medium and the volume needed for all neucleofection samples (including negtive control).
  9. Transfer the volume calculated above of cell suspension into a 50 ml sterile centrifuge tube and spin at 200Xg (800rpm for Beckman Model TJ-6 centrifuge) for 5 min.
  10. Aspirate the supernatant medium completely.
  11. Add Nucleofector solution at the volume of (100 ul X sample number) into the tube and resuspend the cell pellet homogeneously. (Do not let the cells stand in this solution more than 15 min!)
  12. Add 100 ul each of the suspension to the Eppendorf tubes containing plasmid DNA and mix well. Then transfer to the Nucleofector cuvette. Avoid air bubbles in the suspension. Close the cuvette with the blue cap.
  13. Insert the cuvette into the cuvette holder on the Nucleofector II device. Select the appropriate program (U-23 for JAC NDF cells), press "X" key to start the program. (This will take only a few seconds).
  14. Take the cuvette out of the holder and transfer to medium immediately to avoid damaging the cells. Using the plastic pippette provided in the kit, add 500 ul of the pre-warmed growth medium to the cuvette and aspirate back togather with the cell suspension. The cells can stay in the tube for 15 min in the incubator and then transfered to 10 cm tissue culture dish with pre-warmed medium OR put into 10 cm dish directly. Shake the dish forth and back gently to make the cells distribute evenly.
  15. Incubate at 37oC with 5% CO2 and humidity at 95%.
  16. For stable nucleofection, selection drug can be added after 24 hours.

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