The hy pothesis that MEK ERK and PI3K Akt are necessary for the neuronal differentiation and neurite outgrowth of PC12 cells was also tested working with certain inhibitors. Strategies Materials and chemical compounds The fruiting bodies of P. giganteus had been obtained from Nas Agro Farm, Sepang, Selangor, Malaysia. Rat pheo chromocytoma cell line was obtained from American Type Culture Collection two,5 diphenyltetrazolium bromide phosphate buffered saline dimethyl selleck sulf oxide F 12 K medium NGF 7 S from murine submax illary gland, MEK inhibitor and PI3K inhibitor had been obtained from Sigma Co. Fetal bovine serum and horse serum had been purchased from PAA Laboratories Cultivation situation of mushrooms Pleurotus giganteus was maintained on po tato dextrose agar at four 10 C and consistently sub cultured. The substrate formulation for your cultivation of P. giganteus is very similar to that for oyster mushroom cultivation, i. e.
89 94% rubber wood sawdust, five 10% rice bran and 1% calcium carbonate. Polypropylene bags are made use of for substrate bagging and the moisture content inside the substrate was stored at 60% 65%. The temperature for mycelia development, spawn run, and fruiting body formation is 26 32 C. Relative hu midity of 70% and 80 90% throughout mycelia growth and fruiting, respectively, really should be maintained. PP242 molecular weight Direct illu mination should be averted since it has been reported to inhibit the fruiting body formation. A twenty day cycle right after plete colonization with the artificial log is required for every harvest and about 4 harvests could be obtained from every bag of 900 g Cell culture The PC12 cells from ATCC had been maintained in F twelve K medium sup plemented with 2. 5% heat inactivated fetal bovine serum and 15% horse serum with final pH six. eight seven. 2. All incubations have been performed at 37 C in a humidified setting of 5% CO2 and 95% air.
The cells had been maintained inside the logarithmic phase of development and had been subcultured at two three day intervals. For storage, the cells have been frozen at 70 C liquid nitro gen in plete medium supplemented with 5% di methyl sulfoxide as being a cryoprotectant. Extraction of P. giganteus fruiting bodies The fresh fruiting bodies had been sliced, weighed and freeze dried for 1 2 days. The freeze dried fruiting bodies have been then ground using a blender. The resulting dried powder was weighed and kept in four 8 C. Aqueous extraction system was according to Eik et al. Briefly, the freeze dried powder was soaked in distilled water and was left overnight at space temperature and 200 rpm in the shaker. The mix ture was double boiled in water bath for thirty min and fil tered soon after cooling. The resulting aqueous extract was freeze dried and stored at forty C prior to use. For ethanol extraction, the freeze dried powder was soaked in 95% ethanol at space temperature for three days as well as practice was repeated three times.