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.