Cell Preparation

HEK 293T Cells

We optimized conditions for reverse transfection with the HEK 293T cell line. We culture these adherent cells in DMEM containing 10% IFS, 50 units/ml penicillin and 50 µg/ml streptomycin. In this medium, the cells have a doubling time of about 20 hours and we split them every 3-4 days to avoid confluency.

To prepare HEK 293T cells for reverse transfection:

  1. 24 hours before needed, plate 10 x 106 cells in 10 ml medium in a 10 cm dish. Alternatively, 5x106 cells can be cultured for 2 days before transfection. In both cases, cells are still actively growing when harvested.
  2. Within 30-40 minutes before reverse-transfection, in a tissue culture hood harvest cells from the 10 cm dish as follows:

 

  1. Remove medium via vacuum aspiration.
  2. Rinse cells with 2 ml of 4°C trypsin-EDTA (Life Technologies, 25300-054). Allow the solution to spread over the plate and then remove it quickly by vacuum aspiration.
  3. Add 1 ml trypsin-EDTA to the cells and spread it evenly over the plate.
  4. Bang the plate with the palm of your hand to dislodge cells from the surface of the plate.
  5. Place the plate in the tissue culture incubator for 3-5 minutes.
  6. Add 10 ml of 37°C full medium to the plate of cells.
  7. Pipet up and down 12-15 times with a 10 ml pipet until a single cell suspension is obtained. Try to avoid frothing the medium.
  8. Count cells in a hemocytometer.
  9. Aliquot 10 x 106 cells into a sterile 50 ml conical tube.
  10. Bring volume up to 25 ml using warmed full medium.
  11. Close tube and mix by inverting 3-4 times.
  12. Cells are now ready to add to microarrays. If cells are ready before transfection time is over, we keep the 50 ml tube containing the cells in the tissue culture incubator. We have keep cells in the 50 ml tube for up to 30-40 minutes before needing them and have seen no adverse effects. Thus, although it is not our standard practice, cells can be harvested and prepared before beginning transfection of arrays. Always invert cell mix 2-3 times immediately before adding to arrays.

Other Cell Lines

We also tested other adherent cell lines (cos7, NIH 3T3, HeLa, A549) and found them to give transfection efficiencies of 30-50% of HEK293T cells. For these cell lines we also used 10 x 106 cells.

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*Table of Contents *Sabatini Lab *Whitehead Institute for Biomedical Research*