||The 3T3 cell line was established by G. Todaro and H. Green in 1962 from disaggregated Swiss mouse embryos.
||POLYOMAVIRUS; SV40 virus
||Lysophosphatidylcholine (lyso-PC) induces AP-1 activity and c-jun N-terminal kinase activity (JNK1) by a protein kinase C-independent pathway [PubMed: 9153219]
||The cells are contact inhibited.
A confluent monolayer yields 40000 cells/cm2.
The cells should be grown in plastic flasks, they do not grow well on some types of glass surfaces.
A saturation density of approximately 50000 cells/cm2 can be reached.
||Dulbecco's Modified Eagle's Medium (DMEM) modified with 4500 mg/L glucose and bovine calf serum to a final concentration of 10%.
||NNever allow culture to become completely confluent. Subculture when 80% confluent or less.
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 medium, and rinse with PBS without calcium and magnesium.
Remove the solution and add an additional 1 to 2 mL of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37°C) until the cells detach.
Add fresh culture medium, aspirate and dispense into new culture flasks. For 75 cm2 flasks use 4 X 105 cells per flask and subculture every 3 days.
Population Doubling Time 18 hrs
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.
|Subculturing Medium Renewal
||Twice a week
||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).
||Todaro GJ, Green H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17: 299-313, 1963. PubMed: 13985244
Bennicelli JL, et al. Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma. Proc. Natl. Acad. Sci. USA 93: 5455-5459, 1996. PubMed: 8643596
Vogt M, Dulbecco R. Studies on cells rendered neoplastic by polyoma virus: the problem of the presence of virus-related materials. Virology 16: 41-51, 1962. PubMed: 13926482
Todaro GJ, et al. Antigenic and cultural properties of cells doubly transformed by polyoma virus and SV40. Virology 27: 179-185, 1965. PubMed: 4284655
Todaro GJ, et al. Transformation of properties of an established cell line by SV40 and polyoma virus. Proc. Natl. Acad. Sci. USA 51: 66-73, 1964. PubMed: 14104605
Fang X, et al. Lysophosphatidylcholine stimulates activator protein 1 and the c-Jun N-terminal kinase activity. J. Biol. Chem. 272: 13683-13689, 1997. PubMed: 9153219
Chen ST, et al. Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitiis virus by using a modified tetracycline inducible system. Proc. Natl. Acad. Sci. USA 93: 10057-10062, 1996. PubMed: 8816750
Campbell M, et al. The simian foamy virus type 1 transcriptional transactivator (Tas) binds and activates an enhancer element in the gag gene. J. Virol. 70: 6847-6855, 1996. PubMed: 8794326
Hsu DK, et al. Identification of a murine TEF-1-related gene expressed after mitogenic stimulation of quiescent fibroblasts and during myogenic differentiation. J. Biol. Chem. 271: 13786-13795, 1996. PubMed: 8662936
||Banco de Células do Rio de Janeiro