细胞培养技术

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.