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BALB/3T3 clone A31

(ATCC? CCL-163?)

Organism: Mus musculus, mouse / Cell

Type: fibroblast / Tissue: embryo /

Organism Mus musculus, mouse

Tissue embryo

Cell  Type fibroblast

Product Format frozen

Morphology fibroblast

Culture Properties adherent

Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age embryo; 14 to 17 days gestation Strain BALB/c

Applications This cell line is a suitable transfection host.

Storage Conditions liquid nitrogen vapor phase Karyotype modal number = 78; range = 62 to 109.

The stemline number is hypotetraploid. Most of the cells contained only telocentric or acrocentric chromosomes.

Note: Cytogenetic information is based on initial seed stock at ATCC.

Cytogenetic instability has been reported in the literature for some cell lines.

Derivation The BALB/3T3 clone A31 is one of several cell lines (see ATCC CCL-164, BALB/3T12) developed by S.A. Aaronson and G.T. Todaro in 1968 from disaggregated 14- to 17-day-old BALB/c mouse embryos.

Tumorigenic No, the cells were not tumorigenic in immunosuppressed mice, but did form colonies in semisolid medium.

Effects No, in immunosuppressed mice Yes, in semisolid medium

Virus Susceptibility Herpes simplex virus Vesicular stomatitis virus Virus Resistance {265A7263-515F-42F9-89FD-9B140DF21BEC}

Comments The cells are extremely sensitive to contact inhibition of cell division, grow at a high dilution, exhibit a low saturation density and are highly susceptible to transformation in tissue culture by the oncogenic DNA virus, SV40, and murine sarcoma virus Tested and found negative for ectromelia virus (mousepox).

Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: bovine calf serum to a final concentration of 10%.

Subculturing Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. Corning? T-75 flasks (catalog #430641) are recommended for subculturing this product.

Note: Never allow cultures to become completely confluent before subculture. Some important considerations in the handling of 3T3 cells: doubling time is about 18 hours in sparse cultures.

The cells reach a saturation density of about 106 cells per 20 cm2. In order to maintain this property of high contact inhibition, it is necessary to transfer routinely at only high dilutions, otherwise variants tend to be selected having reduced contact inhibition. Such low density makes culture vessels appear sparse and cell growth sensitive to sub-optimal temperature and media conditions.

1，Remove and discard culture medium.

,2，Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.

3，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℃ to facilitate dispersal.

4，Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.

5,Add appropriate aliquots of the cell suspension to new culture vessels.

6,Incubate cultures at 37℃.

Subcultivation Ratio: For 60 mm plates, use an inoculum of 3 x 105 cells per plate and subculture every 3 days. Medium Renewal: Twice per week Cryopreservation Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO Storage temperature: liquid nitrogen vapor phase

Culture Conditions

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37℃

Growth Conditions: The serum used is calf serum, NOT fetal calf serum

. The depositor recommended calf serum because fetal calf serum causes transformation and loss of contact inhibition.