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This increased metastatic cell line was derived using an in vivo selection process of highly metastatic cells from a population of poorly metastatic tumor cells, A375 (ATCC^® No. CRL-1619™) and A375P a donation from from Dr. Isaiah Fidler (Texas MD Anderson Cancer Center, for Set F cells). 

A375P and CRL-1619 were used to create more lung metastases cell lines when injected into mice.The A375-M1 (ATCC^® No. CRL-3222™) cell line was derived by i.v. injection of A375 cells into nude mice. Lung metastases were harvested and amplified in vitro as cell lines. The A375-M1 cell line was reinjected into mice for a second round of selection, lung metastases harvested and amplified in vitro as A375-M2 (ATCC^® No. CRL-3223™) cells. The A375-M1 and A375-M2 cell lines were  transfected with a plasmid containing the ecotropic receptor for murine retrovirus and selected for neomycin resistance. This is useful for RNAse protection assays.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC^® No. 30-2200) or 0.05% (w/v) Trypsin-0.02% EDTA solution (ATCC^® PCS-999-003) 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.
6. Incubate cultures at 37°C. 
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: Every 2 to 3 days

Xu L, et al. Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases. Mol Cancer Res 6:760-769, 2008. PubMed: 18505921

Clark EA, et al. Genomic analysis of metastasis reveals an essential role for rhoC. Nature 406:532-535, 2000. PubMed: 1095231