The 3T3 L1-MBX clone was derived from 3T3-L1 (ATCC CL-173) to ensure close to 100% differentiation to adipocytes and great response to insulin. In addition, 3T3 L1-MBX has a great insulin-stimulated glucose uptake response which is about 8-10 fold window with sub-maximal insulin concentration in 2-deoxyglucose uptake assay (2-DOG). This cell line would be a valuable tool for researchers who are interested in diabetes and obesity research areas.

Note: Never allow culture to become completely confluent.

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

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°C 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.xa0
   The recommended inoculum is 3 to 5 X 10³ cells/cm². Subculture before cultures become 80 to 90% confluent.

Gregoire FM, et al. MBX-102/JNJ39659100, a novel peroxisome proliferator-activated receptor-ligand with weak transactivation activity retains antidiabetic properties in the absence of weight gain and edema. Mol. Endocrinol. 23(7): 975-88, 2009. PubMed: 19389808