two,5 diphenyltetrazolium bromide],phosphate buffered saline,dimethyl 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 have been obtained from PAA Laboratories. Cultivation situation of mushrooms Pleurotus giganteus was maintained on po tato dextrose agar at 4 ten C and regularly sub cultured. The substrate formulation for your cultivation of P. giganteus is related 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 as well as moisture written content during the substrate was stored at 60% 65%. The temperature for mycelia development, spawn run, and fruiting entire body formation is 26 32 C. Relative hu midity of 70% and 80 90% while in mycelia growth and fruiting. respectively, need to be maintained.
Direct illu mination need to be avoided since it has become reported to inhibit the fruiting selleckchem entire body formation. A 20 day cycle just after full colonization in the artificial log is needed for every harvest and about four harvests can be obtained from just about every bag of 900 g. Cell culture The PC12 cells from ATCC were maintained in F 12 K medium sup plemented with two. 5% heat inactivated fetal bovine serum and 15% horse serum with ultimate pH six. eight 7. 2. All incubations have been performed at 37 C within a humidified environment of 5% CO2 and 95% air. The cells had been maintained in the logarithmic phase of growth and were subcultured at two three day intervals. For storage, the cells were frozen at 70 C liquid nitro gen in complete medium supplemented inhibitor canagliflozin with 5% di methyl sulfoxide like a cryoprotectant. Extraction of P. giganteus fruiting bodies The fresh fruiting bodies were sliced, weighed and freeze dried for 1 two days.
The freeze dried fruiting bodies had been then ground applying a blender. The resulting dried powder was weighed and kept in 4 8 C. Aqueous extraction strategy was in accordance to Eik et al. Briefly, the freeze dried powder was soaked in distilled water and was left overnight at room temperature and 200 rpm in the shaker. The mix ture was double boiled in water bath for thirty min and fil tered following cooling. The resulting aqueous extract was freeze dried and kept at forty C just before use. For ethanol extraction, the freeze dried powder was soaked in 95% ethanol at area temperature for three days and also the procedure was repeated three times. The ethanol solvent was evaporated using 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 evaluation. Cell viability and cytotoxicity assay Cell viability and proliferation was determined by MTT assay.