PEA shares the same mechanism of action with other neuroprotectants providing further evidence for the value of kinase signaling in neuroprotection. Calceinacetoxymethyl ester was bought from Alexis Biochemicals or EMD/Calbiochem. Tertbutylhydroperoxide was purchased from Acros Organics. As described previously cell culture The murine hippocampal cell line HT22 was cultured. In short, HT22 cells were developed in Dulbecco s modified Eagle s medium with 1 mM and high glucose E3 ligase inhibitor sodium pyruvate, 5% bovine development serum, 2 mM Glutama and penicillinstreptomycin. Cultures were maintained at a confluency of significantly less than 70% throughout the culturing process. For immunofluorescence investigation, HT22 cells were plated on polyLlysinecoated 12 mm coverslips overnight followed closely by solutions as described in the text. Immunocytochemistry was therefore conducted as described elsewhere in detail. Evaluation of cell viability Oxidative stress was induced by exposing cells to 20 25 M tBHP. In order to determine Organism cell viability in a structure the fluorimetric calcein AM and VYBRANT glucose6phosphate dehydrogenase cytotoxicity assays were conducted in 96 well plates. All 96 well plate assays for HT22 cell viability were done employing a cell density of 2000 cells/well unless noted otherwise. For the calceinAM assay, press was removed from plates after 16 20 hours of tBHP exposure accompanied by replacement with Hank s balanced salt solution with 2 mM CaCl2 and calceinAM dye at a final concentration of 4 M for 20 minutes to load cells. Calcein fluorescence was measured using a fluorimetric plate reader with the right filters. The actual mechanism is that viable cells take up the ester form of calcein and convert it to the nonester form, calcein. Calcein accumulates in viable cells resulting in fluorescence. The VYBRANT G6PD cytotoxicity assays were done 10 12 hours after tBHP coverage based on the manufacturer s directions using a substrate reaction time of 5 6 hours at 37 C and examine at 530 nm excitation and 560 nm emission. In theory, nonviable cells leak their contents into the culture media thus allowing for the assay of enzyme order Avagacestat activity, such as H 6PD activity. All raw data was analyzed, normalized and graphed in Microscoft Excel. Immunocytochemistry after PEA therapy HT22 cells were plated on polyLlysinecoated 12 mm coverslips at 40,000 cells/ml and maintained for 24-hours. The media was eliminated and replaced with media containing 100 M PEA for various time points. After the PEA coverage, the cells were washed and fixed with 401(k) paraformaldehyde followed by immunocytochemistry using polyclonal sera elevated against Akt, pAkt, ERK1/2, phosphoERK1/2, p38 or monoclonal rabbit antiphosphop38 antibody using a technique described elsewhere.