2,5 diphenyltetrazolium bromide],phosphate buffered saline,dimethyl sulf oxide,F twelve K medium,NGF 7 S from murine submax illary gland, MEK inhibitor,and PI3K inhibitor have been obtained from Sigma Co. Fetal bovine serum and horse serum were bought from PAA Laboratories. Cultivation situation of mushrooms Pleurotus giganteus was maintained on po tato dextrose agar at 4 ten C and consistently sub cultured. The substrate formulation to the 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 utilized for substrate bagging and also the moisture content material during 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 development and fruiting. respectively, should be maintained.
Direct illu mination ought to be avoided as it is reported to inhibit the fruiting pan Aurora Kinase inhibitor body formation. A twenty day cycle right after total colonization of the artificial log is required for each harvest and about four harvests could be obtained from every single bag of 900 g. Cell culture The PC12 cells from ATCC were maintained in F 12 K medium sup plemented with 2. 5% heat inactivated fetal bovine serum and 15% horse serum with ultimate pH 6. eight seven. 2. All incubations have been performed at 37 C in the humidified environment of 5% CO2 and 95% air. The cells had been maintained within the logarithmic phase of growth and were subcultured at 2 3 day intervals. For storage, the cells have been frozen at 70 C liquid nitro gen in total medium supplemented find out this here with 5% di methyl sulfoxide like a cryoprotectant. Extraction of P. giganteus fruiting bodies The fresh fruiting bodies have been sliced, weighed and freeze dried for one 2 days.
The freeze dried fruiting bodies were then ground making use of a blender. The resulting dried powder was weighed and stored in four eight C. Aqueous extraction technique 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 after cooling. The resulting aqueous extract was freeze dried and stored at forty C before use. For ethanol extraction, the freeze dried powder was soaked in 95% ethanol at space temperature for 3 days along with the course of action was repeated three times. The ethanol solvent was evaporated making use of a rotary evaporator to present a brownish vis cous extract. Nutritional composition of freeze dried fruiting bodies of P. giganteus Fifty grams sample of P. giganteus fruiting bodies was sent to Consolidated Laboratory Sdn. Bhd. for nutri tional analysis. Cell viability and cytotoxicity assay Cell viability and proliferation was determined by MTT assay.