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This method provides a step-by-step guide to transfecting Drosophila S2 cells with the pRmHA-3 (or similar) vector with insert of choice (in this case SDF-1β-FLAG) and generating a stable cell line. This cell line is then capable of producing the protein of interest under inducible conditions by addition of copper sulfate, which can then be purified and used as desired. This protocol provides an example to finding out when your peak protein production occurs and a method for determining optimal selection conditions.

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[Bio101] Transfection of S2 Cell with DNA Using CellFectin Reagent

Molecular Biology > DNA > Transformation
Author: Darran G. Cronshaw
Darran G. CronshawAffiliation: Center for Autoimmune and Musculoskeletal Diseases, Feinstein Institute for Medical Research, Manhasset, USA
For correspondence:
Bio-protocol author page: a41
3/5/2012, 9542 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.190

[Abstract] This method provides a step-by-step guide to transfecting Drosophila S2 cells with the pRmHA-3 (or similar) vector with insert of choice (in this case SDF-1β-FLAG) and generating a stable cell line. This cell line is then capable of producing the protein of interest under inducible conditions by addition of copper sulfate, which can then be purified and used as desired. This protocol provides an example to finding out when your peak protein production occurs and a method for determining optimal selection conditions.

Materials and Reagents

  1. Drosophila Schneider 2 (S2) cells (Life Technologies, Invitrogen™, catalog number: R690-07)
  2. Dros-SFM (Life Technologies, Invitrogen™, catalog number: 10797-025)
  3. pHASβAG (pRmHA-3 vector with SDF-1β-FLAG inserted. Contains a metallothionein promoter inducible with Cu2+)
  4. pUC-Hsneo (contains the neomycin-resistant cassette required for stable selection)
  5. CellFectin reagent (Life Technologies, Invitrogen™, catalog number: 10362-010)
  6. FBS (Life Technologies, Invitrogen™, catalog number: 10437-028)
  7. Penicillin-Streptomycin (P/S) (Life Technologies, Invitrogen™, catalog number: 15140-122)
  8. G418 sulphate (Life Technologies, Invitrogen™, catalog number: 11811-031)
  9. CuSO4 (Sigma Aldrich, catalog number: C8027)
  10. HT supplement (Life Technologies, Invitrogen™, catalog number: 11067-030)
  11. Transfection solutions (see Recipes)

Equipment

  1. 6-well plate (Corning Incorporated, catalog number: 353046)
  2. 24-well plate (Corning Incorporated, catalog number: 353047)

Procedure

  1. Drosophila Schneider 2 (S2) cells are plated out at 1.6 x 106 cells/well (6-well plate, 2 ml /well) and incubated overnight at room temperature (RT).
    Note: The optimal temperature of S2 cells is actually 28 °C and they do not need CO2 to culture.
  2. Following day, cells are washed 2x with Dros-SFM (no FBS, P/S or HT) 1 ml/well.
  3. Prepare the ‘DNA mix’ and ‘CellFectin Mix’ transfection solutions.


  4. 100 μl /sample (i.e. 400 μl total) of the DNA mix is added to 100 μl /sample (i.e. 400 μl total) CellFectin mix. Solution is gently mixed and left at RT for 30 min.
  5. After 30 min, 800 μl /sample (3.2 ml total) of Dros-SFM (no FBS, P/S or HT) is added to the DNA/CellFectin mix.
  6. 1 ml of this solution is then added to the appropriate wells containing the S2 cells and incubated at RT for 5 h.
  7. Following the 5 h incubation, medium is aspirated off and Dros-SFM + 10% FBS + P/S + HT (= complete media) is added to each well (2 ml/well).
  8. Plates are incubated at RT for 48 h.
  9. At 48 h post-transfection the media is removed and replaced with 2 ml media containing G418 sulphate at indicated concentrations. The cells are transferred to 24-well plates (one 6-well ≥ eight 24-wells) supplemented with additional media + G418 to 1 ml.

  10. Cells are incubated at RT with media changed every 5 days (1 ml/well).
  11. After 3 weeks, untransfected cells should be killed by the G418 at least at the highest concentration. The transfected cells should be fine and healthy.
  12. Cells within the 4 μl and 8 μl CellFectin groups are pooled and split 1:1 into two 24-well plates (i.e. 8 wells /plate). After 1 h, media is removed and replaced with either complete SFM + 1.5 mg/ml G418 or complete SFM + 1.5 mg/ml G418 + 1 mM CuSO4.

  13. Cell lysates are taken at 2, 3, 4 and 5 days post-CuSO4 addition to determine peak time of SDF-1β-FLAG production.

Recipes

  1. Transfection solutions
    a.  DNA mix (per sample):                        Total (9 samples)
         4 μg pHASβAG = 1.67 μl                   15.0 μl
         0.2 μg pUC-Hsneo = 0.63 μl                5.7 μl
         100 μl Dros-SFM (no FBS or P/S)         900 μl
    b.  CellFectin Mix (per sample):                Total (4 samples)
         4 μl or 8 μl CellFectin reagent             16 or 32 μl
         100 μl Dros-SFM                               400 μl

References

  1. Bunch, T. A., Grinblat, Y. and Goldstein, L. S. (1988). Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16(3): 1043-1061.


How to cite this protocol: Cronshaw, D. G. (2012). Transfection of S2 Cell with DNA Using CellFectin Reagent. Bio-protocol Bio101: e190. DOI: 10.21769/BioProtoc.190; Full Text



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