The primary (Mel-270) and metastatic (OMM-2.5) UM cell lines, which have been characterized extensively,31 (link),32 (link) were kindly provided by Martine J. Jager (Leiden University Medical Center, Leiden, The Netherlands). Cells were grown under normoxic conditions at 37°C with 5% CO2 in UM medium and passaged twice weekly by trypsinization.
For the experiments, cells at passages 21 to 29 were collected by trypsinization for 2 to 3 minutes, seeded on to 8-well slides (Sarstedt, Nümbrecht, Germany, 104 cells/well), 96-well plates (8.103 cells/well), 48-well plates (8.104 cells/well), or 6-well plates (2.105 cells/well) and allowed to attach overnight. For the treatments involving serum deprivation, cells were washed twice for 10 minutes with serum-free medium before introducing the test medium. Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water. Cells were incubated in normal UM medium with 10% fetal bovine serum (FBS) or serum free medium ± adiponectin (30 µg/mL) for 1 day.
For the experiments, cells at passages 21 to 29 were collected by trypsinization for 2 to 3 minutes, seeded on to 8-well slides (Sarstedt, Nümbrecht, Germany, 104 cells/well), 96-well plates (8.103 cells/well), 48-well plates (8.104 cells/well), or 6-well plates (2.105 cells/well) and allowed to attach overnight. For the treatments involving serum deprivation, cells were washed twice for 10 minutes with serum-free medium before introducing the test medium. Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water. Cells were incubated in normal UM medium with 10% fetal bovine serum (FBS) or serum free medium ± adiponectin (30 µg/mL) for 1 day.
