Representaciones de mundo/ Símbolos culturales

Preparation of Biopsy M aterial

In the operating theatre, tum our specim ens w ere im m ediately placed in sterile 30ml universals (Scientific Laboratory Supplies, N o ttin g h a m ) containing H am s FIG m edia (Invitrogen Ltd., Inchinan, R enfrew shire) su p p lem en ted w ith kanam ycin (50^g/m l), p en ic illin /stre p to m y c in ( 1 OOlU/ m l/1 OOjLfg / ml) and am photericin B (2.5jug/ml). Sam ples w ere processed im m ediately u p o n arrival in the laboratory, although they rem ained viable for up to 5 days in the transport m edia. V enous blood sam ples w ere collected in sterile 30ml universals containing EDTA an d stored at -70°C.

All handling of the biopsy and blood sam ples was carried out in a Class 11 lam inar air flow cabinet (ICN Pharm aceuticals, Costa Mesa, CA). T he biopsy was rem oved from the universal containing transport m edia an d placed in a sterile petri dish (Helena Biosciences, Sunderland, UK).

A sam ple of the biopsy w eighing approxim ately 25mg was rem oved for DNA extraction. As m uch tissue as possible was frozen dow n in LN^ to allow for subsequent extractions. The rem aining tissue (approxim ately lOmg) was sliced using crossed sterile scalpels (size 10 blades) u n til it was fine enough to be pipetted. The m inced tissue was th en pipetted into a sterile universal and 2ml H am s FIO culture m edia, containing 10% foetal calf serum (FCS) (Labtech International, Sussex), and 1ml collagenase solution (Sigma Aldrich, Gillingham, Dorset) w ere added. The tissue was incubated at 37°C to allow the tissue structure to be broken dow n by th e action of the collagenase. After 30 m inutes incubation, 7ml culture m ed ia (Hams FIO + 10% FCS) w as added to the universal and the sam p le centrifuged for 5 m inutes at lOOOrpm. The su p ern atan t w as rem oved an d the sam ple resuspended in 10ml culture m edia and transferred to a 25cm^ tissue culture flask (Triple Red, Thame, Oxfordshire). The sam ple was

T haw ing Frozen Cell Stocks

A sm all beaker was filled w ith water at approxim ately 37°C. A vial of cells w as rem oved from LN^ and placed into the beaker to thaw . The cells w ere pipetted out of the vial and slowly allowed to drip dow n the side of a 25cm^ flask containing 9ml media. The cells w ere th en incubated at 37°C and the m edia changed after 24 hours to allow the cells tim e to attach to the flask.

Feeding Cells.

Cells w ere fed once a w eek or w hen the m edia h ad changed from dark pin k to yellow in colour, depending on w hich was sooner. The absorption of n u trien ts from the m edia by the cells results in the presence of w aste products w hich tu rn the m edia acidic and cause the m edia to becom e yellow in colour. The m edia was aspirated from the flasks using a sterile glass pipette and replaced w ith new media: 10ml for 25cm^ flasks; 20ml for 75cm^ flasks and 35ml for 150cm^ flasks.

Passaging Cells

Cells were passaged w hen they had reached confluence, in the follow ing way:

1 X 25cm^ flask transferred into 1 x 75cm^ flask

1 X 75cm^ flask split into 3 x 75cm^ flask

1 X 75cm^ flask transferred into 1 x 150cm^ flask

1 X 150cm^ flask split into 3 x 150cm^ flask

The old m edia was aspirated using a sterile glass pipette. The cells w ere w ashed twice w ith sterile Hanks Buffered Saline Solution (HESS) (Invitrogen). In order to detach the cells from the bottom of the flask, 3m l of trypsin solution (Sigma Aldrich) were added to the flask and the cells incubated at 37°C for 15-30 m inutes. To inactivate the trypsin, 7ml of m edia were added to the cells and the flask contents pipetted into a sterile

universal. The cells were then centrifuged at lOOOrpm for 5 m inutes. T he supernatant w as rem oved and the cells resuspended in 10m l of media. Each tim e the cells were treated w ith trypsin solution the passage n u m b e r

was increased by 1.

Freezing Cells

Any cells that were not required for experim entation w ere frozen. A 0.4m l sam ple of the cell suspension was added to 19.6ml of Isoton II (C ounter (Beckman Coulter UK Ltd., H igh Wycombe, Bucks) and the cells co unted using a C oulter C ounter (Beckman Coulter). The rem aining cells w ere centrifuged at lOOOrpm for 5 m inutes and the su p ern atan t rem oved before the cell pellet was resuspended in FCS containing 10% d im eth y l sulphoxide (Sigma Aldrich) to a final concentration of 1 m illion cells per 1ml. The cell suspension was pipetted into a cryovial (1ml suspension per vial) and stored at -70°C for 24 hours. The frozen cells w ere th e n transferred to LN2 storage tanks.