Fresh medium containing increasing three different concentrations of the GNPs (10, 50, and 100 μl) was added to each well and the cells were incubated for 6, 24, 48, and 72 h time periods.
Then, cells that grew exponentially in the present of 20 nM of BPR0L075 were obtained and subsequently cultured in complete medium containing increasing concentrations of the drug to 30 nM of BPR0L075.
A mixed population of cells that grew exponentially in the present of 15 nM of BPR0L075 were obtained and further cultured in complete medium containing increasing concentrations of the drug to 20 nM of BPR0L075.
After overnight growth at 37°C under rotating agitation, the bacterial suspension was used to initiate cultures in 10 ml of SS medium containing increasing concentrations of nicotinate or MgSO4.
Briefly, KB cells were initially incubated in completed medium containing 5 nM of BPR0L075 that yielded 40% cell survival for a period of 3∼4 weeks, and the cells that proliferated were repeatedly subcultured in completed medium containing increasing concentrations of the drug (at 20% increment each time).
After digestion, the cells were resuspended in modified Eagle's medium containing increasing Ca2+ concentrations (200 μM, 500 μM, and 1 mM).
This was replaced by medium containing increasing doxycycline (0 100 μM) to generate a standard curve, against which the mouse serum samples were analysed.
Plating medium was replaced with DMEM containing 1% 4× charcoal-stripped serum for 48 hr, and then with treatment medium containing increasing concentrations of BPS or E2 (10 15 to 10 7 M) alone or BPS concentrations plus 10 9 M E2.
Confluent cells were washed with DMEM-F12 medium and stimulated separately overnight with fresh culture medium containing increasing concentrations of TNF (0, 0.01, 0.1, 1 and 10 ng ml−1).
