NIH/3T3



BCRJ code:
0191
Cell Line:
NIH/3T3
Species:
Mus musculus

Vulgar Name: Mouse
Tissue:
Embryo
Cell Type:
Fibroblast
Morphology:
Fibroblast
Disease:
Normal
Growth Properties:
Adherent
Age Ethinicy:
embryo
Applications:
This cell line is a suitable transfection host.
The NIH/3T3 cell line is highly sensitive to sarcoma virus focus formation and leukemia virus propagation and has proven to be very useful in DNA transfection studies. [PubMed: 222457]
Virus Succeptibility:
Murine leukemia virus , Murine leukemia virus
Biosafety:
1
Culture Medium:
Dulbecco's Modified Eagle's Medium (DMEM) modified to contain 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate and bovine calf serum 10%.
Subculturing:
DO NOT ALLOW THE CELLS TO BECOME CONFLUENT Subculture at least twice per week at 80% confluence 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 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: Twice per week
Subcultivation ratio: Inoculate 3 to 5 X 103 cells/cm2
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:
Jainchill JL, et al. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol. 4: 549-553, 1969. PubMed: 4311790

Andersson P, et al. A defined subgenomic fragment of in vitro synthesized Moloney sarcoma virus DNA can induce cell transformation upon transfection. Cell 16: 63-75, 1979. PubMed: 84715

Copeland NG, Cooper GM. Transfection by exogenous and endogenous murine retrovirus DNAs. Cell 16: 347-356, 1979. PubMed: 222457

Loffler S, et al. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus. J. Virol. 71: 42-49, 1997. PubMed: 8985321

Berson JF, et al. A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus tyep 1 strains. J. Virol. 70: 6288-6295, 1996. PubMed: 8709256

Jones PL, et al. Tumor necrosis factor alpha and interleukin-1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB. Mol. Cell. Biol. 17: 6970-6981, 1997. PubMed: 9372929

Gonzalez Armas JC, et al. DNA immunization confers protection against murine cytomegalovirus infection. J. Virol. 70: 7921-7928, 1996. PubMed: 8892915

Siess DC, et al. Exceptional fusogenicity of chinese hamster ovary cells with murine retrovirus suggests roles for cellular factor(s) and receptor clusters in the membrane fusion process. J. Virol. 70: 3432-439, 1996. PubMed: 8648675

Jang SI, et al. Activator protein 1 activity is involved in the regulation of the cell type-specific expression from the proximal promoter of the human profilaggrin gene. J. Biol. Chem. 271: 24105-24114, 1996. PubMed: 8798649

Medin JA, et al. Correction in trans for Fabry disease: expression, secretion, and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector. Proc. Natl. Acad. Sci. USA 93: 7917-7922, 1996. PubMed: 8755577

Lee JH, et al. The proximal promoter of the human transglutaminase 3 gene. J. Biol. Chem. 271: 4561-4568, 1996. PubMed: 8626812

Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc. Natl. Acad. Sci. USA 93: 136-140, 1996. PubMed: 8552591

Cranmer LD, et al. Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins. J. Virol. 70: 7929-7939, 1996. PubMed: 8892916

Shisler J, et al. Induction of susceptibility to tumor necrosis factor by E1A is dependent on binding to either p300 or p105-Rb and induction of DNA synthesis. J. Virol. 70: 68-77, 1996. PubMed: 8523594

Cavanaugh VJ, et al. Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism. J. Virol. 70: 1365-1374, 1996. PubMed: 8627652

Westerman KA, Leboulch P. Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc. Natl. Acad. Sci. USA 93: 8971-8976, 1996. PubMed: 8799138

The NIH/3T3, a continuous cell line of highly contact-inhibited cells was established from NIH Swiss mouse embryo cultures in the same manner as the original random bred 3T3 (ATCC CCL-92) and the inbred BALB/c 3T3 (ATCC CCL-163). The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays.
Depositors:
Wanderley de Souza - Universidade Federal do Rio de Janeiro
ATCC:
CRL-1658