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The 3T3 MEFs KO cell line (ATCC CRL-2753) is homozygous for a disruption of the caveolin-1 gene Cav-1 (-/-) while the 3T3 MEFs WT cell line (ATCC CRL-2752) is Cav-1 (+/+). Analysis of cultured fibroblasts from Cav-1 null embryos reveals a loss of caveolin-2 protein expression; defects in the endocytosis of a known caveolar ligand, (fluorescein isothiocyanate-albumin); and a hyperproliferative phenotype. These phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. RefRazani B, et al. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J. Biol. Chem. 276: 38121-38138, 2001. PubMed: 11457855

A culture deposited with the ATCC in September of 2002 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cyclin. The cells were assayed for mycoplasma, by the Hoechst stain, PCR and the standard culture test, after a six-week period following treatment. All tests were negative.

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 pipetting gently.
5. Add appropriate aliquots of cell suspension to new culture vessels.
6. Incubate culture vessels at 37°C.

Razani B, et al. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J. Biol. Chem. 276: 38121-38138, 2001. PubMed: 11457855

Sotgia F, et al. Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells. Mol. Cell. Biol. 22: 3905-3926, 2002. PubMed: 11997523