Comparative Studies on the In Vitro: MATERIALS AND METHODS(3)
The pellet was resuspended in 30% Percoll solution and overlaid on top of a 60% Percoll solution. After centrifugation at 800 X g for 30 min, the cells that sedimented at the 3060% interface were collected. The cells were washed twice in PBS supplemented with DNase and BSA. The cells were then resuspended in phenol red-free RPMI-1640 supplemented with 0.1 mM sodium pyruvate, 100 U/ml penicillin, 100 ^g/ml streptomycin, and 10% fetal calf serum depleted of steroids by treatment with dextran-coated charcoal. buy ortho tri-cyclen online
Cells were seeded at 1 X 105 cells/cm2 in 75-cm2 tissue culture flasks and incubated overnight at 37°C and 5% CO2. The medium was changed the next day and subsequently every 3 days. At confluence, cells were trypsinized (trypsin/ EDTA) and subcultured into 12-well tissue culture plates. The medium was changed every 3 days. Cell purity was assessed by immunocytochemistry using antibodies against cytokeratin (Dako, Carpenteria, CA), vimentin (Zymed, San Francisco, CA) and factor VIII (Dako). The purity of the stromal cell preparations used in these studies was > 97%.