Er (Molecular Devices, Sunnyvale, CA, USA). Data were expressed as the
Er (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 Behavior Pattern/MSC I/class 1-2 M/class 3 C/class 4-a bV-10 n ( ) 0 (0) 0 (0) 2 (17) 10 (83)PETL-10 p-value GABA-1 p-value n ( ) n ( ) 0 (0) 0 (0) 10 (83) 2 (17) 0.000a 0.000b 0.c0 (0) 0 (0) 12 (100) 0 (0)0.000a 0.000b 0.000cLipid peroxidation was assessed by measuring malondialdehyde (MDA) in extracts of PC12 cells using a lipid peroxidation assay kit (Cayman Chemical, Ann Arbor, MI, USA). This kit works on the principle of condensation PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27906190 of one molecule of either malondialdehyde (MDA) or 4-hydroxyalkenals with two molecules of N-methyl-2phenylindole to yield a stable chromophore. MDA levels were assayed by measuring the amount produced by 5 ?105 cells. A absorbance at 500 nm was determined using an ELISA reader (spectraMAX 340, Molecular Devices, Sunnyvale, CA, USA).Assay of PGE2 concentration and Caspase-3 ActivationFisher’s exact test. Pearson’s chi-square test: all seizure classes taken together. c Kendall’s tau-c: all seizure classes taken together. I: Initial (class 1-2). M: middle (class 3). C: critical. PETL-10: Pu-Erh Leaf extract, 10 mg/kg. GABA-1: gamma-aminobutyric acid, 1 mg/kg. V-10: vehicle control, with normal saline.seizure Imatinib (Mesylate) site patterns in the SE mice compared with the vehicle (Table 1, GTL and GABA, p < 0.001,).Protection from KA toxicityPGE2 release and caspase-3 activity were measured by ELISA assay. PC12 cells (5 ?105) were added to 0.5 ml homogenization buffer (0.1 M phosphate pH 7.4, 1 mM EDTA) and homogenized. The lysate was then centrifuged at 12,000 ?g for 15 min at 4 . The supernatant was transferred to a clean test tube, and its total protein content was analyzed using the Bradford assay (Bio-Rad, Hemel, Hempstead, UK). PGE2 concentration and caspase-3 activity were determined using PGE 2 and caspase-3 ELISA kits (R D Systems, Minneapolis, MN, USA). A absorbance at 450 nm was determined using a microplate reader (spectraMAX 340, Molecular Devices, Sunnyvale, CA, USA).Statistical analysisWe further evaluated H E stained section of the brains of KA-stressed FVB mice. KA (10 mg/kg) caused epilepticus and neuronal d.
