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After six generations the stopping criterion was reached and a growth medium was obtained which produced sixteen times more enzyme than the starting medium.
RB-DP performs on a comparable level to the other two algorithms for small allowed tardiness levels and then exceeds the performance of MBFD starting medium levels of request lifetimes and then clearly exceeds both algorithms with the higher levels starting 400 time units.
The extracted palatal and molar mucosal tissues or minced tails were placed on a 0.1% gelatin-coated 30-mm tissue culture dish and maintained in MF-start medium (Toyobo, Osaka, Japan) at 37°C with 5% CO2.
Plat-E cells, which were also used to produce retroviruses, were maintained in DMEM containing 10% FBS, 50 unitsml−1 penicillin, 50 µgml-1 streptomycin, 1 µgml−1 puromycin, and 10 µgml−1 blasticidin S. To establish TTFs, the tails from adult mice were peeled, minced into 1-cm pieces, placed on culture dishes, and incubated in MF-start medium (Toyobo) for 5 days.
The cultures were grown in cotton-stoppered baffled shake-flasks with starting medium volume of 10% of the flask volume.
The oxygen contents in the starting medium were normalized assuming an O2 concentration of 217 μM in air-saturated medium at 37 °C (n=3).
When the differentiation period started, medium was changed at 64 h (day 3) and at 216 h (day 10; Fig. 5a).
To ensure that the starting medium was consistent between experiments, aliquots (10 μl) were washed, derivitized, and analyzed by GC-MS (aboveove) with each experiment.
Alveolar bone cells, seeded at a density of 1 × 10 cells cm−2 in six-well plates, were cultured in the starting medium described above, in the presence of 0 1 μg ml−1 of P. gingivalis LPS.
In the first step, a growth phase, with 2 L starting medium containing 35 g L-1 of lactose as carbon source, 27°C and pH regulated at 4.8 (with 6 M ammonia) was conducted.
