Passaging cells

Passaging cells

1. Pour out media from flasks.
2. Wash with Hanks. 5 ml per flask.
3. Tilt around then dump.
4. Add 4 ml of Trypsin / EDTA to each flask. Tip, then bang.
5. Add 20% FBS NCTC media and tilt. 4 ml per flask.
6. Scrape (most of the small cells are already released; proliferative cells lift up easy, differentiative cells are more adherent).
7. Place contents into a 50 ml Falcon tube.
8. Spin at 1000 RPM for 5 min (#5 setting = 1000)
9. Dump off supernatant.
10. Add enough media to allow 2 ml of cells per flask (RCHO-cells start to differentiate at day two. At that time a flask contains ca. 1 million cells. They are spread by 1 : 3).
11. Each flask should have 8 (or 10) ml of 20% FBS NCTC media + 2 ml of cells for a total of 10 (or 12) ml.