T 37 , and treated with 150 M KA for 60 or 120 min. Cells were then washed twice at room temperature with Hank’s balanced salt solution (HBSS without phenol red). Cellular fluorescence was monitored on a Fluoroskan Ascent fluorometer (Labsystems Oy, Helsinki, Finland) using an excitation wavelength of 485 nm and emission wavelength of 538 nm.MTT reduction assay for cell viabilityThe Rat pheochromacytoma cell line PC12 was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 (v/v) fetal bovine serum, 5 horse serum, 100 U/ml penicillin and 100 g/ml streptomycin at 37 in a humidified incubator under 5 CO 2. Confluent cultures were passaged by trypsinization. Cells were washed twice with warm DMEM (without phenol red), then treated in serum-free medium. In all experiments, cells were treated with GABA and/or KA-stress for the indicated times.Preparation of cell extractsTest medium was removed from culture dishes and cells were washed twice with ice-cold phosphate-buffered saline, scraped off with the aid of a rubber policeman, and centrifuged at 200 ?g for 10 min at 4 . The cell pellets were resuspended in an appropriate volume (4 ?10 7 cells/ml) of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10 g/ml aprotinin, and 5 g/ml pepstain A. The suspension was then sonicated. Protein concentration was determined by Bradford assay (BUdRMedChemExpress BRDU BioRad, Hemel, Hempstead, UK) after cells were suspended to 2 mg/ml with in lysis buffer.Western PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25112874 blottingCell viability was measured using blue formazan that was metabolized from colorless 3-(4,5-dimethyl-thiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial dehydrogenases, which are active only in live cells. PC12 cells were preincubated in 24-well plates at a density of 5 ?10 5 cells per well for 24 h. Cells incubated with various concentrations of GABA were treated with 150 M KA for 24 h, and grown in 0.5 mg/ml MTT at 37 . One hour later, 200 l of solubilization solution was added to each well and absorption values read at 540 nm on microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA). Data were expressed as the mean percent of viable cells vs. control.Lactate dehydrogenase (LDH) release assayCytotoxicity was determined by measuring the release of LDH. PC12 cells treated with various concentrations of GABA were incubated with 150 M KA for 24 h and the supernatant was then assayed for LDH activity. A absorbance was read at 490/630 nm using a microtiter plate reader. Data were expressed as the mean percent of viable cells vs. 150 M KA control.Calcium release assayProtein samples containing 50 g of protein were separated on 12 sodium dodecyl sulfate polyacrylamidePC12 cells with various concentrations of GABA were treated with 150 M KA for 24 h and the supernatantHou Journal of Biomedical Science 2011, 18:75 http://www.jbiomedsci.com/content/18/1/Page 4 ofwas used to assay the release of Ca2+. The 10 l supernatant was added to 1 ml Ca2+ reagent (Diagnostic Systems, Holzheim, Germany) and mixed well, allowed to stand for 5 min, then transferred the 100 l supernatant to 96 well. Calcium concentration was determined using a microplate reader with a 620 nm absorbance and quantified with a 10 mg/ml Ca2+ standard solution.Measurement of lipid peroxidationTable 1 Effects of Pu-Erh tea leaf extract and GABA on the predominant behavior patterns/maximal seizure class (MSC) and 10-h mortality rate of the mice with 5-hour KA-induced SEVariables Mortality B.
