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Basal release of Etn metabolites was monitored by taking 30 μl aliquots of medium at T=0, 1, 2 and 3 h for scintillation counting (below) and by replacing medium removed with fresh R0 to maintain volume.
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Seeded M109 cells were washed several times with fresh medium to remove unattached cells, then culture medium was removed with aspiration.
Excess cells and medium were removed with PBS and calcein-labeled tumor cells (2 × 10/100 μl) were added in serum-free α-MEM medium supplemented with 0.01% BSA.
Culture medium was removed with the help of a micropipette and the biofilm was washed gently with PBS as described above.
The medium was removed with syringe.
After 9 days of incubation at 18°C in the dark, the MMN medium was removed with a sterile pipette.
Then, 1 mL release medium was removed with a precision pipette and sterile tips at 2, 4, 6, 24, 48, and 96 hours.
Medium was removed with a 24 channel comb and 20 μl of 1 4 diluted CellTiterGlo reagent was added for 20 min.
Cells were allowed to adhere for 72 h and non-adherent cells were removed with medium changes.
Non-adherent cells were removed with the medium and experiments were started.
The erythrocytes and lymphocytes were removed with the medium change after 3 4 days.
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