Thus, to determine neuronal damage, find protocol the medium was aspirated and kept at 4 C until analysis. The plated neurons were Inhibitors,Modulators,Libraries lysed by three freeze thaw cycles with 1 ml HEPES buffer containing 0. 02% Triton X 100. The lysates were also kept at 4 C for analysis. Before the assay, both intracellular and extracellu lar fractions were separated by centrifugation for 10 min utes at 14,000 rpm in a microcentrifuge at 4 C. The pellets were discarded, and the supernatants were used to measure LDH activity as follows. Samples of these supernatants were diluted with 0. 5 ml of Tris NaCl buffer pH 7. 2 at 30 C. Reactions were started by adding 2. 5 ml of 0. 244 mmol l NADH into the Tris NaCl buffer solution. Absorbance was measured at 340 nm, and the decrease in absorbance was followed every 0.
5 seconds for 2 minutes, the slope of the decrease showed the LDH activ ity. The percentage of LDH leakage was calculated using the ratio between extracellular LDH activity and the sum of intracellular and extracellular LDH activity, and results were expressed as percentage of control values. Determinations Inhibitors,Modulators,Libraries were performed in triplicate for each sample, and Inhibitors,Modulators,Libraries the results averaged. Single cell calcium imaging This was carried out Inhibitors,Modulators,Libraries essentially as described previously, using Fura 2 acetoxymethyl ester , a membrane permeable and calcium sensitive radiometric dye, to fluorimetrically measure variations in the intracellu lar free calcium concentration by monitoring its ratio of absorption at 510 nm upon excitation at 380 nm or 340 nm. Briefly, hippocampal neurons, plated onto cover slips, were loaded with 5 umol l Fura 2 Amol l and 0.
02% pluronic acid F 127 for 30 minutes in Krebs buffer supplemen ted with 0. 1% fatty acid free BSA, at 37 C in an incubator in a atmosphere of 95% CO2 5% O2. After washing Inhibitors,Modulators,Libraries three times with Krebs buffer to remove excess probe, coverslips were placed in a superfusion chamber on the stage of an inverted fluorescence microscope. Hippocampal neurons were alter nately excited at 340 and 380 nml using an optical splitter,and the emitted fluorescence was captured through a 40�� oil objective connected to a digital camera. Acquired images were pro cessed using MetaFluor software. The areas of the cell bodies were drawn, and the average value of pixel intensities was evalu ated at each time point. Image acquisition was performed every second for a total of 35 minutes.
Results were expressed by plotting the time course of the ratio of fluores cence intensity emitted at 510 nm after excitation alternately at 340 and 380 nm. All of the compounds tested were prepared in Krebs buffer and added to the cultured neurons by superfusion using a rapid pressurization system in 95% O2 5% CO2. The basal ratio Rapamycin was measured for the first 2 minutes of the experiment, before the stimuli were made.
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