Vulgar Name: Human
|Tissue:||Mammary Gland/Breast; Derived From Metastatic Site: Pleural Effusion|
|Age/Ethinicy:||43 YEARS OLD; Caucasian|
|Applications:||This cell line is suitable as a transfection host.|
|DNA Profile:||Amelogenin: X CSF1PO: 12 D13S317: 11,12 D16S539: 9 D5S818: 9,12 D7S820: 9,12 THO1: 8,9 TPOX: 8,11 vWA: 17|
|Tumor Formation:||Yes, in nude mice; forms poorly differentiated adenocarcinoma|
|Additional info:||No virus particles. Ultrastructural features include microvilli and desmosomes, glycogen granules, large lysosomes, bundles of cytoplasmic fibrils. The SK-BR-3 cell line overexpresses the HER2/c-erb-2 gene product.|
|Culture Medium:||McCoy's 5A Medium is modified to contain 1.5 mM L-glutamine and 2200 mg/L sodium bicarbonate and fetal bovine serum to a final concentration of 10%.|
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Remove and discard culture medium.
Briefly rinse the cell layer with PBS without calcium and magnesium to remove all traces of serum which 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.
Incubate cultures at 37°C.
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.
Medium Renewal: 2 to 3 times per week
Subcultivation ratio: 1:2 is recommended
|Culture Conditions:||Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37°C|
|Cryopreservation:||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).|
|References:||Fogh J. Human tumor cells in vitro. New York: Plenum Press; 1975. Trempe GL. Human breast cancer in culture. Recent Results Cancer Res. 57: 33-41 , 1976. PubMed: 1013510 Fogh J, et al. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J. Natl. Cancer Inst. 58: 209-214, 1977. PubMed: 833871 Fogh J, et al. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J. Natl. Cancer Inst. 59: 221-226, 1977. PubMed: 327080 Pollack MS, et al. HLA-A, B, C and DR alloantigen expression on forty-six cultured human tumor cell lines. J. Natl. Cancer Inst. 66: 1003-1012, 1981. PubMed: 7017212 Hudziak RM, et al. Monoclonal antibodies directed to the Her2 receptor. US Patent 5,677,171 dated Oct 14 1997 Littlewood-Evans AJ, et al. The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma. Cancer Res. 57: 5386-5390, 1997. PubMed: 9393764 Chavany C, et al. p185erbB2 binds to GRP94 in vivo. J. Biol. Chem. 271: 4974-4977, 1996. PubMed: 8617772|
|Depositors:||MARIZE CAMPOS VALADARES;UFG|