Peter van Mourik

71 Forskolin-induced swelling protocol C. Noggin conditioned medium (NCM) production Note: HEK293T Noggin hFc cells are poorly adhesive cells (especially the first passage after thawing). At the first passage after thawing leave the cells for at least 5 days without touching. Make sure to handle the cells gently during washing with PBS0 to avoid detaching. An extra passaging step before starting the harvesting steps is strongly recommended to let the cells recover from the freeze-thawing step. 1. Thaw a cryovial with HEK293T Noggin hFc cells by putting 13 mL of pre-warmed DMEM +/+ in a 15 mL tube (when thawing multiple cryovials, use a 50 mL tube with 30 mL DMEM +/+). 2. Transfer the cells from the cryovial to the 15 mL tube by resuspending the cells with DMEM +/+. 3. Centrifuge the 15 mL tube at 450 g for 5 min. Remove the supernatant and resuspend the cell pellet in 40 mL pre-warmed DMEM +/+ with additional G418 (final concentration 500 µg/mL). 4. Make sure that a homogeneous cell suspension is achieved and transfer the cell suspension to a T175 flask. 5. Incubate the T175 flask for 7 days at 37 °C and 5% CO 2 . 6. Split the cells when 90-100 % confluence is achieved into 6 x T175 flasks. 7. Gently remove the DMEM +/+ from the T175 flask. 8. Gently wash the cells once with 5 mL PBS0 and discard the PBS0. 9. Add 1.5 mL 0.05 % Trypsin EDTA, leave at 18-23 ° C for 7 - 10 min until the cells are detached. 10. Add 10.5 mL DMEM +/+ medium, take up all the cells and resuspend until a homogenous cell suspension is achieved. 11. Transfer the cell suspension to 15 mL tube 12. Count the cells (with e.g. hemocytometer) 13. Add 5*10 6 cells in 25 mL culture medium and transfer to a new T175 flask: o 1 x T175 in culture medium with G418 (500 µg/mL), use this flask for the next batch (usually cells are confluent after 3 - 4 days). o X (depending on amount of cells) x T175 in culture medium without G418. When the cells seeded in DMEM +/+ without G418 are ~90% confluent (usually after 3 - 4 days): 14. Remove the DMEM +/+ from the T175 flasks 15. Gently (to avoid detachment of the cells) add 50 mL of Ad-DF+++ per flask. 16. Incubate the flasks at 37 °C and 5% CO 2 for 8-10 days. 4