Calciumphosphate transfection and generation of stably transfected ...

Buffer 2xHBS:
50mM HEPES
1,5mM Na 2HPO 4
280mM NaCl
-> pH 7,1 w/ NaOH, sterile filter, aliquot
Calciumphosphate transfection
and generation of stably transfected S2-cell line
Day 1:
Seed Schneider-2 cells in Schneider’s S-2-Medium supplemented w/ 10% FCS into 6well-plates
at the density required.
Incubate O/N at 27°C.
Day 2:
Optional: Change medium 3-4 h prior to transfection
Transfection (amount for 1 well of a 6-well plate (diameter 35mm)
Tube A:
9 µl 2M CaCl2
5 µg DNA (expression plasmid/pCoBlast 1:20)
-> fill up to 75µl w/ H 2O
Tube B: 75 µl 2xHBS
Slowly add content of tube A to tube B (dropwise, ca. 2min)
Incubate the mixture (precipitate is forming) for 30 min at RT
Add the solution dropwise to the medium containing cells.
Incubate O/N at 27°C.
Day 3:
Scrape cells off the bottom of the 6-Well plate and transfer into a vial.
Centrifuge 2min at 1000 rcf.
Aspire supernatant (discard) and resuspend cells in 1xPBS
Centrifuge 2min at 1000 rcf.
Aspire supernatant completely (discard)
Optional: Assign cells to two (or more) selection conditions (we used to do it in duplicate)
Resuspend cells in 3ml S2-Medium supplemented w/ 25 µg/ml Blasticidin and 50 µg/ml
Blasticidin
Seed cells into the same wells, from which they were taken (in order not to lose
transfected cells sticking to the bottom; may be dispensable)
Cells lacking pCoBlast will die.
Day 5:
Medium change
Aspire cells with medium, centrifuge 2 min at 1000rpm, discard supernatant.
Resuspend cells in 50% conditioned Schneider’s S2 medium supplemented with the
respective concentration of Blasticidin S (3 ml) and reseed cells into the respective well.

Example 25 µg/ml Blasticidin S: 50% fresh medium + 50% conditioned medium + 25 µg/ml
final concentration Blasticidin.
To obtain conditioned medium, grow wildtype Schneider 2 cells in T175 culture bottles. For
splitting, aspire cells with medium, centrifuge 2 min at 1000 rpm. Save supernatant. Pass
supernatant through a 0,25 µm sterile filter fitted onto a 50 ml syringe.
Weeks 2 and 3:
Perform medium changes with 50% conditioned selective medium every 2-3 days.
Pass all cells of one well into a T25 bottle when cells reach 5x10 6 /ml
Clone picking is not usually performed.
Trick:
Add selective medium to cells remaining in the 6-well-plate. Let them continue to grow. At a
density of ~ 5x10 6 /ml split cells into 9 wells of a 12 well plate, let grow for 1-2 days. Add serial
dilutions of CuSO 4 from 0,01 – 2 mM CuSO 4 and incubate cells for ~24 h. Prepare cell extracts
with RIPA buffer, assay ~40 µg of total protein by immunoblotting.
Use part of the cells to prepare cryo-stocks w/ DMSO.
Expand remaining cells up to 3x175 cm 2 -bottles, transfer the cells to a 500 ml-spinner
flask (27°C, 116 rpm) and add the same volume of fresh medium (w/o Blasticidin).
Propagate cells into larger spinner flasks to keep the volume/surface ratio small. Induce
desired volume of cells w/ CuSO 4 using the concentration determined before (see above).

Trouble shooting:
Heavy metal ions may be lost into the medium from some glass spinner flasks which
may induce MT-promoter driven expression. Use plastic lab ware (polycarbonate) to
overcome this problem.
Leaky expression may also become apparent as a result of high copy numbers of the
respective construct. Vary the ratio of pCoBlast and pMTagIt.