n part, by activation . Furthermore, PS-341 Proteasome inhibitor PS-341 Proteasome inhibitor we provide evidence that this effect of PEA is not mediated through the activation of CB2. The results of the present study identify PEA as a potential therapeutic agent for the treatment of neurodegenerative diseases in which oxidative stress occurs. Furthermore, PEA shares a similar mechanism of action with other neuroprotectants providing further evidence for the importance of kinase signaling in neuroprotection. Palmitoylethanolamine, JWH 015, AM 1242 and AM 630 were purchased from Alexis Biochemicals. Calcein acetoxymethyl ester was purchased from Alexis Biochemicals or EMD/Calbiochem. Tert butylhydroperoxide was purchased from Acros Organics.
The murine hippocampal cell line HT22 was cultured as described previously.
In brief, HT22 cells were grown in Dulbecco,s modified Eagle,s medium with high glucose and 1 mM sodium pyruvate, 2 mM Glutamax, 5% bovine growth serum and penicillin streptomycin. Cultures were kept at a confluency of PS-341 179324-69-7 less than 70% during the culturing process. For immunofluorescence analysis, HT22 cells were plated on poly L lysinecoated PS-341 179324-69-7 12 mm coverslips overnight followed by treatments as described in the text. Immunocytochemistry was subsequently conducted as described elsewhere in detail. Oxidative stress was induced by exposing cells to 20 25 M tBHP.
The fluorimetric calcein AM and VYBRANT glucose 6 phosphate dehydrogenase cytotoxicity assays were conducted in 96 well plates in order to assess cell viability in a high throughput format.
All 96 well plate assays for HT22 cell viability were conducted using a cell density of 2,000 cells/well unless noted otherwise. For the calcein AM assay, media was removed from plates after 16 20 hours of tBHP exposure followed by replacement with Hank,s balanced salt solution with 2 mM CaCl2 and calcein AM 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 appropriate filters. The underlying mechanism is that viable cells take up the ester form of calcein and convert it to the non ester form, calcein. Calcein accumulates in viable cells resulting in elevated fluorescence.
The VYBRANT G 6 PD cytotoxicity assays were conducted 10 12 hours after tBHP exposure according to the manufacturer,s instructions with a substrate reaction time of 5 6 hours at 37 and read at 530 nm excitation and 560 nm emission.
In principle, non viable cells leak their contents into the culture media thus allowing for the assay of enzyme activity, such as G 6 PD activity. All raw data was analyzed, normalized and graphed in Microscoft Excel. HT22 cells were plated on poly L lysine coated 12 mm coverslips at 40,000 cells/ml and maintained for 24 hours. The media was removed and replaced with media containing 100 M PEA for various time points. After the PEA exposure, the cells were rinsed and fixed with 4% paraformaldehyde followed by immunocytochemistry using polyclonal sera raised against Akt, pAkt, ERK1/2, phospho ERK1/2 , p38 or monoclonal rabbit anti phospho p38 antibody using a method described elsewhere. After completion of ICC and mounting, images were acquired at 20× magnification using an Olympus IX70 fluorescence microscope. TIFF images were analyzed