Osteoblast differentiation experiment: BMP-2 induced osteogenesis in mouse MC3T3 cell line

MC3T3 cells clone 1b were grown in alpha-MEM with 10% fetal calf serum (FCS, Gibco), 1% penicilin/streptomycin (Pen/Strep) and 1% L-Glutamine (L-Glu). For differentiation experiment, cells were seeded on 6 cm dishes (3x105cells/dish in 5 ml medium) and grownt to confluence for 3 days at 37C / 5% CO2. Cells were then stimulated with osteogenic stimulus (10 mM beta-glycerophosphate (GP, Sigma), 50 µM ascorbic acid (AA, Wako) and 1 mg/ml BMP-2 (Novartis)), or with 10mM GP alone for 1 or 3 days. Control cells-day 0, medium. Experiment was done in triplicate. Total RNA was extracted, treated with DNAse, and purified according to the manufacturer’s protocol (RNeasy mini kit, QIAGEN). RNA samples were analyzed on oligonucleotide microarrays Affymetrix GeneChip® Murine Genome U74Av2. Keywords: ordered