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.

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. Ref 

Tested and found negative for ectromelia virus (mousepox).

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.

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 which 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¡ãC 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¡ãC.

DO NOT ALLOW THE CELLS TO BECOME CONFLUENT Subculture at least twice per week at 80% confluence or less.

Subcultivation Ratio: Inoculate 3 to 5 X 103 cells/cm2

Medium Renewal: Twice per week

Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

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

Temperature: 37¡ãC

Growth Conditions: The serum used is important in culturing this line. Calf serum is recommended and not fetal bovine serum. The calf serum initially employed and found to be satisfactory was from the Colorado Serum Co. Denver.