The MC3T3-E1 (mouse pre-osteoblast) cell line was obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Science and grown in α-minimal essential medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). To induce mineralization, MC3T3-E1 cells were treated for different time points (1, 3, 5, 7, 14 and 21 days) with osteogenic medium (OM), which was composed of complete medium supplemented with 50 µg/ml ascorbic acid (Sigma-Aldrich; Merck KGaA), 10 mM β-glycerophosphate (Sigma-Aldrich; Merck KGaA) and 50 ng/ml BMP2 (Novus Biologicals, LLC) in a humidified incubator at 37°C under 5% CO2. The medium was changed every 3 days. To validate the role of the PI3K/AKT signaling pathway in osteogenic differentiation, 15 mM PI3K inhibitor (LY294002; Selleck Chemicals) was added to the OM (4°C for 3 days) and the medium changed every 3 days.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.