||LNCaP clone FGC
||Prostate; Derived From Metastatic Site: Left Supraclavicular Lymph Node
||Adherent, Single Cells And Loosely Attached Clusters
||LNCaP clone FGC was isolated in 1977 by J.S. Horoszewicz, et al., from a needle aspiration biopsy of the left supraclavicular lymph node of a 50-year-old Caucasian male (blood type B+) with confirmed diagnosis of metastatic prostate carcinoma.
||This cell line is suitable as a transfection host.
||human prostatic acid phosphatase; prostate specific antigen
||These cells are responsive to 5-alpha-dihydrotestosterone (growth modulation and acid phosphatase production).
The cells do not produce a uniform monolayer, but grow in clusters which should be broken apart by repeated pipetting when subcultures are prepared.
They attach only lightly to the substrate, do not become confluent and rapidly acidify the medium.
Growth is very slow.
The cells should be allowed to incubate undisturbed for the first 48 hours after subculture.
When flask cultures are shipped, the majority of the cells become detached from the flask and float in the medium. Upon receipt, incubate the flask (in the usual position for monolayer cultures) for 24 to 48 hours to allow the cells to re-attach. The medium can then be removed and replaced with fresh medium.
If desired, the contents of the flask can be collected, centrifuged at 300 X g for 15 minutes, resuspended in 10 mL of medium and dispensed into a single flask.
||RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate and 10% of fetal bovine serum.
||Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. T-75 flasks are recommended for subculturing this product.
Remove and discard culture medium.
Briefly rinse the cell layer with with PBS without calcium and magnesium to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Maintain cultures at a cell concentration between 1 X 10e4 and 2 X 10e5 cells/cm2.
Incubate cultures at 37°C.
Population Doubling Time: about 34 hours
NOTE: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 6th edition, published by Alan R. Liss, N.Y., 2010.
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
|Thawing Frozen Cells:
||SAFETY PRECAUTION: Is highly recommend that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that
some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris.
1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. For cells that are sensitive to DMSO is recommended that the cryoprotective agent be removed immediately. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes.
4.Discard the supernatant and Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio).
5. Incubate the culture in a appropriate atmosphere and temperature (see "Culture Conditions" for this cell line).
NOTE: It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
||Models for prostate cancer. 37New York: Liss; 1980; Gibas Z, et al. A high-resolution study of chromosome changes in a human prostatic carcinoma cell line (LNCaP). Cancer Genet. Cytogenet. 11: 399-404, 1984. PubMed: 6584201
||MARIA DE FATIMA MAGALHAES LAZARI; INFAR- UNIFESP