Three drops cell suspension were placed in a single well of a standard 12 well culture plate. The cells were allowed to adhere for two hours at 37 C, then 1 ml maintenance medium was added to each well. Geneticin or puromy cin pressure was maintained during chondrogenesis. Micro masses were cultured in the maintenance med ium http://www.selleckchem.com/products/Calcitriol-(Rocaltrol).html containing an ITS premix and 5 ug ml human transferrin for two weeks. The mineralization phase was induced using a MEM medium containing 5% fetal bovine serum, ITS premix, 5 ug ml human transferrin and 7 mM beta glycerolphosphate from Day 14 until Day 21. Each condition was performed in tripli cate. Total RNA from micro masses was isolated after 7, 14 or 21 days in culture using the Nucleospin RNA II kit.

Protein extraction of the micro masses stably overex pressing FRZB or controls after seven days was per formed using cell extraction buffer supplemented Inhibitors,Modulators,Libraries with 1 mM phenylmethanesulfonyl and 5% protease inhibitor cocktail, followed by quantification using the Pierce BCA Protein Assay kit. Some ATDC5 micro masses were fixed in 95% ice cold methanol for staining. For Picrosirius Red, Inhibitors,Modulators,Libraries micro masses were stained for one hour in Picrosirius Red in a saturated aqu eous solution of picric acid washed three times with 0. 5% acetic acid in water and air dried. For Safranin O, micro masses were stained for one hour in Safranin O washed three times with water and air dried. Quantifica tion of the staining was performed by dissolving the micro masses with 1 M NaOH or 6M Guanidine HCl and by measuring the absorbance at 540 and 512 nm respectively with the Infinite M200.

cDNA synthesis and Quantitative Real Time PCR Complementary Inhibitors,Modulators,Libraries DNA was synthesised from 1 ug of RNA isolated from tibia articular cartilage and For TaqMan assays analysis was performed using the PerfeCTa qPCR FastMix UNG using the following conditions, 1 minute at 95 C, 40 cycles of 3 seconds of denaturation at 95 C, followed by 20 seconds of annealing extension at 60 C. All experiments were performed in duplicate. For SYBRgreen, quantitative analysis was performed as follows, 10 minutes at 95 C, 40 cycles of 15 seconds of denaturation at 95 C, fol Inhibitors,Modulators,Libraries lowed by 60 seconds of annealing extension at 60 C. Melting curve analysis was performed to ensure amplifi cation of a specific product. The Corbett Rotor Gene 6000 was used for both systems.

Results are expressed using the comparative threshold method and were normalised to housekeeping gene Hprt. Mouse rib chondrocyte isolation and proliferation analysis Rib and sternum chondrocytes were isolated from three six week old wild type and three Inhibitors,Modulators,Libraries Frzb mice, as described with minor modifications. The sternum buy inhibitor was longitudinally cut, followed by complete removal of the ventral part of the ribcage. The ribcage was washed three times in Dulbeccos phosphate buffered saline with 1% AB.