2,five diphenyltetrazolium bromide],phosphate buffered saline,dimethyl sulf oxide,F twelve K medium,NGF seven S from murine submax illary gland, MEK inhibitor,and PI3K inhibitor have been obtained from Sigma Co. Fetal bovine serum and horse serum had been purchased from PAA Laboratories. Cultivation ailment of mushrooms Pleurotus giganteus was maintained on po tato dextrose agar at four ten C and often sub cultured. The substrate formulation for the cultivation of P. giganteus is 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 applied for substrate bagging as well as the moisture material inside the substrate was stored at 60% 65%. The temperature for mycelia growth, spawn run, and fruiting physique formation is 26 32 C. Relative hu midity of 70% and 80 90% for the duration of mycelia development and fruiting. respectively, needs to be maintained.
Direct illu mination needs to be prevented because it is reported to inhibit the fruiting hop over to here physique formation. A twenty day cycle right after complete colonization within the artificial log is required for each harvest and about 4 harvests is often 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 two. 5% heat inactivated fetal bovine serum and 15% horse serum with last pH 6. 8 seven. two. All incubations were carried out at 37 C in a humidified setting of 5% CO2 and 95% air. The cells were maintained while in the logarithmic phase of development and had been subcultured at two 3 day intervals. For storage, the cells have been frozen at 70 C liquid nitro gen in comprehensive medium supplemented order Dinaciclib with 5% di methyl sulfoxide as a cryoprotectant. Extraction of P. giganteus fruiting bodies The fresh fruiting bodies have been sliced, weighed and freeze dried for 1 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 8 C. Aqueous extraction method 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 inside a shaker. The combine ture was double boiled in water bath for 30 min and fil tered just after cooling. The resulting aqueous extract was freeze dried and stored at 40 C before use. For ethanol extraction, the freeze dried powder was soaked in 95% ethanol at room temperature for 3 days plus the system was repeated 3 instances. The ethanol solvent was evaporated working with a rotary evaporator to provide a brownish vis cous extract. Dietary 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 established by MTT assay.