Calciumphosphate transfection and generation of stably transfected ...

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).