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After 5 hours, the medium was change to DMEM without FBS.
Then the medium was change to HCO3−-free or 25 mM HCO3− DMEM/F12 and incubated for 15 min. In some experiment groups, 10 µM CFTRinh172 (Sigma), 10 µM KH7 (ChemDiv), 50 ng recombinant FSH were added to the media.
After 24 h, medium was change to EBM2 supplemented medium (Lonza) with 2% FBS (foetal bovine serum, Sigma-Aldrich) and 1×105 mBM-VPC from Dll4+/−, Dll4GOF and respective WT, were put over cultured HUVEC monolayer.
Then the medium was change to HCO3−-free or 25 mM HCO3− DMEM/F12 and incubated for 30 min. In some experiment groups, 10 µM CFTRinh172 (Sigma) or/and 50 ng recombinant FSH were added to the media.
Six hours after transfection, the culture medium was change to DMEM supplemented with 1% FCS.
Medium was change every other day and dishes were monitored daily for cellular outgrowth.
Similar(53)
Culture medium was changed every other day.
The medium was changed every 24 h.
Growth medium was changed every 3 days.
The medium was changed twice a week.
Cell culture medium was changed every day.
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