04/10/2018
- One vial of Schwann cells (primary culture) is thawed in 1 T75 flask coated with gelatin 0,15 %
- Growing medium:
- Schwann cell medium (commercial, from ScienCell)
- SCGS (commercial supplements, from ScienCell) 1%
- FBS 12’5 %
- Penicillin-Streptomycin 1 %
*Every time that the medium is changed, is necessary to add NRG1-B1 and Forskolin to the medium –> 10 ml medium + 1 microl NRG1-B1 + 2 microl Forskolin.
10/10/2018
- Expanding:
- Remove the medium and wash the cells gently with PBS 1x
- Add trypsin-EDTA 0’25%
- Stop the trypsinisation with growing medium
- Replant the cells at 1:3 (from 1T75 –> 3 T75)
15/10/2018
- Freezing:
- Freeze the cells in an special frozen medium: Cryostore (3 million cells per vial)
- Coating in coverslips (to ICC):
- 5 plates (60 mm) with 14 coverslips (12 mm) –> coated with Poly-D-Lysine 1/40 (in Borate Buffer) for 1 h at 37ºC
- Seed 250000 cells/plate
