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Top agar containing cells was poured onto the base agar and the plates were incubated at 37 °C/5% CO2 and media (0.5 ml) were replenished every 3 days.
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At every designated interval, buffer solutions (4 mL) in the beaker were taken out and fresh buffer solution (4 mL) was replenished to keep a constant volume.
At designated intervals, the solution of AH (4 mL) in the beaker was taken out and a corresponding fresh solution (4 mL) was replenished to keep a constant volume.
At each given time point, 1 ml released solution were collected and were replenished with equal volume of PBS solution.
After culturing for 72 hr, the cells were replenished with 1.5 mL of fresh media containing 4 μg/mL CTB (Sigma) (i.e., 6 μg per plate).
After culturing for 72 hr, the cells were replenished with 1.5 mL of fresh media containing 4 μg/mL CTB (Sigma) (i.e., 6 μg per plate) and kept at 4°C for 1.25 hr.
First, 8-day-differentiated 3T3-L1 adinocytes in six-well tissue cultissuelates were replenished with 3 ml of DMEM (Dulbecultureodified Eagle's medium) suplatesnted were 10% calf sereplenished Bio-Products) per with and incubated for 8 h at 37°C prior to addition of adiponectin.
Primary cultures were replenished with 1 ml of fresh human interleukin-2 (IL-2 -containing IL-2 -containingserum medIL-2 -containing on days 5, 10 and 15, and were stinactivatedth the sautoserums peptide (50 μg mediumand with autologus PBMC (1×10 cells ml−1), which had been inactivated with 50 μg ml−1 of mitomycin C at 37°C for 30 min, as antigen-presenting cells, on day 10.
Culture media were replenished every 2 days after DIV 9 by replacing 1 mL of the conditioned media with 1 mL of fresh growth medium that lacked rat serum but was supplemented with glucose (10 mM).
Decimated companies were replenished by fresh troops.
Water glasses were replenished frequently.
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