Growing and splitting the cells

The human prostatic carcinoma cell lines PC3, DU145, JCA1, ALVA31, and LNCaP can be cultured in standard RPMI complete medium (composition see below). Usually, 400,000 to 500,000 cells are seeded in 12 ml of RPMI complete medium in a regular T75 flask and incubated at 37°C in 7% CO₂ (5% CO₂ works, too) for 3 to 4 days when the cells are about 80% confluent. Then, the cells are detached from the surface by trypsinization (with 5 ml of 0.1% trypsin and 0.04% EDTA, Sigma T-3924). The cells are washed once with RPMI and reseeded in a new flask.

Preparation of frozen stocks in liquid nitrogen

Resuspend the cells in FCS (HyClone SH30070.03) containing 10% DMSO at about 2,000,000 cells/ml. Distribute 1 ml aliquots of the cell suspension into cryo-vials, and place the vials in an isopropanol-filled cryo-container (e.g. Nalgene). Keep the cryo-container over night in a -80°C freezer, then transfer the cryo-vials to liquid nitrogen for long-term storage.

How to bring frozen cells back into culture

Wash the defrosted cells (about 2,000,000 cells) once in RPMI complete medium, then seed all cells in 12 ml of RPMI in a T75 flask and incubate as described.

Standard RPMI complete medium - Composition

To a 500 ml bottle of RPMI-1640 (GibcoBRL 11875-093) add:

• 5.0 ml of a Penicillin(10,000 U)-Streptomycin(10 mg)-L-Glutamine(200 mM) solution (Sigma G-1146)
• 5.0 ml of Sodium Pyruvate (100 mM, Sigma S-8636)
• 5.0 ml of 1 M HEPES (pH 7.0)
• 0.5 ml of 2-Mercaptoethanol solution (50 mM, preparation descibed below)
• 50 ml of heat inactivated Fetal Calf Serum (HyClone SH30070.03, heat inactivate for 40 min at 56 degrees Celsius)

Preparation of 2-ME solution

Add 50 microliters of 2-mercaptoethanol to 14.2 ml of RPMI (serum-free), sterile filtration, store in aliquots at -20 degrees Celsius.

Concentrations of antibiotics for the selection of stable transfectants

Values in the table indicate the concentration of the corresponding antibiotic for the different cell lines. The values in brackets indicate the volume of antibiotic stock solution to add to 12 ml RPMI complete medium, when G418=geneticin stock (10 mg/ml, Sigma G-1279), puromycin stock (50 µg/ml, Sigma P-7255), and hygromycin stock (20 mg/ml, Calbiochem 400051).

Cell line		G418		puromycin		hygromycin
ALVA31		400 µg/ml (480 µl)		0.5 µg/ml (120 µl)		100 µg/ml (60 µl)
PC3		100 µg/ml (120 µl)		0.5 µg/ml (120 µl)		120 µg/ml (72 µl)
DU145		400 µg/ml (480 µl)		0.5 µg/ml (120 µl)		300 µg/ml (180 µl)
JCA1		200 µg/ml (120 µl)
LNCaP		200 µg/ml (240 µl)				200 µg/ml (120 µl)