The hy pothesis that MEK ERK and PI3K Akt are needed for your neuronal differentiation and neurite outgrowth of PC12 cells was also tested utilizing certain inhibitors. Procedures Supplies and chemicals The fruiting bodies of P. giganteus had been obtained from Nas Agro Farm, Sepang, Selangor, Malaysia. Rat pheo chromocytoma cell line was purchased from American Sort Culture Collection two,5 diphenyltetrazolium bromide phosphate buffered saline dimethyl Cyclopamine Hedgehog inhibitor sulf oxide F twelve K medium NGF seven S from murine submax illary gland, MEK inhibitor and PI3K inhibitor had been obtained from Sigma Co. Fetal bovine serum and horse serum were bought from PAA Laboratories Cultivation problem of mushrooms Pleurotus giganteus was maintained on po tato dextrose agar at 4 ten C and on a regular basis sub cultured. The substrate formulation for your cultivation of P. giganteus is comparable to that for oyster mushroom cultivation, i. e.
89 94% rubber wood sawdust, 5 10% rice bran and 1% calcium carbonate. Polypropylene bags are used for substrate bagging along with the moisture information in the substrate was stored at 60% 65%. The temperature for mycelia development, spawn run, and fruiting physique formation is 26 32 C. Relative hu midity of 70% and 80 90% during mycelia growth and fruiting, respectively, really should be maintained. selleck chemicals Dinaciclib Direct illu mination needs to be averted as it is reported to inhibit the fruiting physique formation. A twenty day cycle soon after plete colonization of your artificial log is required for every harvest and about four harvests might be obtained from each and every bag of 900 g Cell culture The PC12 cells from ATCC have been maintained in F twelve K medium sup plemented with 2. 5% heat inactivated fetal bovine serum and 15% horse serum with last pH 6. eight 7. two. All incubations have been performed at 37 C inside a humidified environment of 5% CO2 and 95% air.
The cells have been maintained while in the logarithmic phase of development and had been subcultured at two 3 day intervals. For storage, the cells were 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 were sliced, weighed and freeze dried for one 2 days. The freeze dried fruiting bodies had been then ground working with a blender. The resulting dried powder was weighed and stored in four eight C. Aqueous extraction strategy was according to Eik et al. Briefly, the freeze dried powder was soaked in distilled water and was left overnight at area temperature and 200 rpm in the shaker. The combine ture was double boiled in water bath for thirty min and fil tered immediately 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 area temperature for 3 days as well as the procedure was repeated 3 times.