Culture of Human Prostatic Carcinoma Cell Lines
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:
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).
|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)|