Reagents

• Medium: 500 ml Dulbecco’s Modified Eagle Medium (Gibco #41966-029) 55 ml FCS (10 %) 2.8 ml Gentamycin Solution (Sigma G-1272, 10 ml))

• TrypsinTrypsin (10x) lyophilised (Gibco 25095-019)

• SolutionVersene 1:5000 (Gibco 15040-033, 100 ml) Take 2 bottles (2X100 ml) of Versene and pipet 10 ml of each bottle into the bottle with the lyophilised Trypsin to redisolve it. Then redistribute 10 ml of the Trypsin solution back into each Versene bottle.

• PBS

Procedures

Starting from a Kryotube: Prewarm the medium at 37°C and fill two culture flasks (25 ml for 150 cm2 , 5 ml for 25 cm 2 ). Rapidly thaw the cells (at 37°C) and distribute them in two concentrations in the flasks. Change the medium after 12 hrs or once the cells have attached.

Splitting Cells: Aspirate the medium from the flask. Wash the cells carefully with PBS to remove residual medium. Add 1-2ml of Trypsin Solution (equilibrated at RT) to the flask (150 cm2 ) and incubate at 37°C until cells have detached (1-2 minutes). Prepare a new flask with fresh medium. Block trypsinization by adding a few ml of medium. Take a fraction of the cell solution and inocculate the new flask. Typically, when splitting confluent HEK 293 cells in a 1:10 ratio, confluency is reached again after 2-3 days.