Passaging cells Pour out media from flasks. Wash with Hanks. 5 ml per flask. Tilt around then dump. Add 4 ml of Trypsin / EDTA to each flask. Tip, then bang. Add 20% FBS NCTC media and tilt. 4 ml per flask. Scrape (most of the small cells are already released; proliferative cells lift up easy, differentiative cells are more adherent). Place contents into a 50 ml Falcon tube. Spin at 1000 RPM for 5 min (#5 setting = 1000) Dump off supernatant. 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). 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.