In brief, 1,500 cells were plated in 96 well plates on day 1 for 24 hours in 100 ul of MEM con taining 10% FBS, and then starved on day 2 with 100 ul of MEM containing 0. 1% FBS for 24 hours. On day 3, cells were stimulated for 96 hours with vehicle or with different concentrations of MSU in 100 ul of MEM containing 10% FBS. After 96 selleck Palbociclib hours, 20 ul of CellTiter 96 Aqueous One Solution Reagent were directly added to the culture wells. Cells in the presence of the reagent were further incubated for 3 hours at 37 C in a 5% CO2 humidified atmosphere, and then the absorbance Inhibitors,Modulators,Libraries was recorded at 490 nm. The quantity of formazan product corresponding to the optical density at 490 nm absorb ance is directly proportional to the number of living cells in culture.
Confocal microscopy Confluent OBs were stained with 2 uM CMTMR and then stimulated with 0. 5 mg of MSU for 48 hours at 37 C. Confocal microscopy analyses were performed with Olympus Inhibitors,Modulators,Libraries Fluoview 300 microscope by using differential interference contrast and helium neon lasers, magnification 400. Evaluation of mineralization Mineralization of cell cultures was evaluated by alizarin red S staining. OBs were seeded at 2 105 cells well in six well tissue culture dishes and maintained in MEM, 10% FBS supplemented with 10 mM B glycerophosphate, at 37 C in a humidified atmosphere containing 5% CO2. Culture medium was replaced every 3 days until day 20. OBs were treated with MSU Inhibitors,Modulators,Libraries or ve hicle at day 8. At day 20, cells were fixed for 20 minutes with buffered formalin and then stained for 20 minutes with 40 mM ARS, pH 4. 0 to 4.
2 at room temperature. After four washes Inhibitors,Modulators,Libraries with distilled H2O, ARS was extracted, as previously described. In brief, ARS cells were incubated 30 minutes with acetic acid and then heated 10 minutes at 85 C, pH was re stored at 4. 2 with NaOH, and ARS absorbance was read at 405 nm. MMP activity Evaluation Inhibitors,Modulators,Libraries of generic matrix metalloproteinases was assessed with the SensoLyte Generic MMP assay kit that detects the activity of a variety of MMPs, including MMP 1, 2, 3, 7, 8, 9, 12, 13, and 14. Five FAM and QXL520, labeled FRET peptide substrates, were used for continuous measurement of the enzyme activities. On the cleavage of the FRET peptide by MMPs, the fluorescence of 5 FAM was recovered and monitored at excitation emission wavelengths of 490 nm 520 nm. Confluent OBs were treated 24 hours with or without 0.
5 mg MSU selleck catalog in MEM containing 1% FBS. Medium was then centrifuged 2 minutes at 10,000 rpm, and 50 ul of supernatant was added to 50 ul of MMP substrate for 20 minutes. MMP activity in MSU stimulated cells was compared with MMP activity in untreated cells. RNA isolation and real time PCR OB total RNA was isolated by using Trizol. In brief, around 106 confluent cells, stimulated with MSU or vehicle, were washed in PBS and then homogenized in 1 ml Trizol. Total RNA was then extracted, according to the manufacturers protocol.