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Medium changes replaced two-thirds of the culture medium.
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The next day, complete medium was changed and replaced by complete medium with 1.5 mM of calcium or normal medium that contains 0.06 mM of calcium.
The medium was changed and replaced with fresh medium containing MSNs in next day.
After 6 hours incubation, the medium was changed and replaced with incomplete medium to remove dead cells.
One day after, medium was changed and replaced with fresh culture medium, containing 10% serum and 1 ng/mL catumaxomab (Neovii, Fresenius Biotech) and increasing concentrations of sEpCAM (0.2 - 5 ng/mL).
After 4 h, the medium was changed and replaced by serum-free Neurobasal medium supplemented with 2% B27 and 1 mM GlutaMax (Invitrogen) after 24 h.
Following overnight culture in 2% FBS, culture medium was changed and replaced with 0.25% FBS for 24 hr to synchronize the cells.
Medium was replaced by fresh supplemented medium.
Culture medium was replaced 72 hr later and changed medium twice weekly thereafter.
Medium was replaced 30 min after plating, and half the medium changed every 5 7 days.
Medium was replaced 60 min after plating, and half the medium changed every 5 7 days.
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