||This cell line is a suitable transfection host.
||The cells contain an activated N-ras oncogene.
||Dulbecco's Modified Eagle's Medium (DMEM) with 1% non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate, 1.0 g/L glucose, 90%; fetal bovine serum, 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. T-75 flasks are recommended for subculturing this product.
Remove and discard culture medium.
Briefly rinse the cell layer 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.
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, 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).
||Chen TR, et al. Intercellular karyotypic similarity in near-diploid cell lines of human tumor origins. Cancer Genet. Cytogenet. 10: 351-362, 1983. PubMed: 6652615
Geiser AG, et al. Suppression of tumorigenicity in human cell hybrids derived from cell lines expressing different activated ras oncogenes. Cancer Res. 49: 1572-1577, 1989. PubMed: 2647289
Rasheed S, et al. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 33: 1027-1033, 1974. PubMed: 4132053
Adams RA, et al. Direct implantation and serial transplantation of human acute lymphoblastic leukemia in hamsters, SB-2. Cancer Res. 28: 1121-1125, 1968. PubMed: 4872716
Proc. Am. Assoc. Cancer Res. 8: 1, 1967.
Hu M, et al. Purification and characterization of human lung fibroblast motility-stimulating factor for human soft tissue sarcoma cells: identification as an NH2-terminal fragment of human fibronectin. Cancer Res. 57: 3577-3584, 1997. PubMed: 9270031
Iida A, et al. Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system. J. Virol. 70: 6054-6059, 1996. PubMed: 8709228
Brenneman M, et al. Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases. Proc. Natl. Acad. Sci. USA 93: 3608-3612, 1996. PubMed: 8622983
Seiffert D. Hydrolysis of platelet vitronectin by calpain. J. Biol. Chem. 271: 11170-11176, 1996. PubMed: 8626663
Hocking AM, et al. Eukaryotic expression of recombinant biglycan. J. Biol. Chem. 271: 19571-19577, 1996. PubMed: 8702651
||Bryan Eric Strauss, Universidade de São Paulo