||Mammary Gland/Breast; Derived From Metastatic Site: Pleural Effusion
||The MDA-MB-468 cell line was isolated from a pleural effusion of a 51-year-old Black female patient with metastatic adenocarcinoma of the breast.
||This cell line is a suitable transfection host.
||Although the tissue donor was heterozygous for the G6PD alleles, the cell line consistently showed only the G6PD A phenotype. There is a G -> A mutation in codon 273 of the p53 gene resulting in an Arg -> His substitution. EGF receptor is present at 1 X 10e6 per cell.
||Leibovitz's L-15 Medium contains 2 mM L-glutamine and no sodium bicarbonate and fetal bovine serum to a final concentration of 10%.
Note: The L-15 medium formulation was devised for use in a free gas exchange with atmospheric air. A CO2 and air mixture is detrimental to cells when using this medium for cultivation.
||Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Remove and discard culture medium.
Briefly rinse the cell layer 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.
||Atmosphere: air, 100%
||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).
||1206: Brinkley BR, et al. Variations in cell form and cytoskeleton in human breast carcinoma cells in vitro. Cancer Res. 40: 3118-3129, 1980. PubMed: 7000337
22429: Siciliano MJ, et al. Mutually exclusive genetic signatures of human breast tumor cell line
||MARILIA MEIRA DIAS; UNIVERSIDADE ESTADUAL DE CAMPINAS