It is well known that treatment with peroxides depletes GSH levels in RPE cells leading to apoptosis

Samples were fixed with 3.7% paraformaldeide, permeabilized with 1:100 NP-40 and stained with Alexa-488 23237488 phalloidin which binds to actin filaments. Ganglioside M1 rafts were detected on NK cells treated with fluvastatin upon engagement of different surface receptors at 37uC with the specific antibody followed by GAM and staining with cholera toxin subunit A conjugated with Alexafluor488. In some experiments, NK cells cultured as described were stained with anti-perforin mAb and anti-FasL mAb or anti-calnexin mAb 16580199 as a marker of endoplasmic reticulum or anti-CD107a mAb followed by anti-isotype specific Alexafluor488 or 647 GAM. Analysis was performed with the confocal microscope FV500, Olympus IX81, equipped with oil objectives PlanAPO 40x and 60x NA 1.40 and images were taken with the computer program FluoView 2.1. Images were analyzed with the analysis FIVE computer program to enumerate the % of NK cells with rafts or granule containing FasL and/or perforin. Cholesterol Quantification Highly purified ex-vivo NK cells were incubated with fluvastatin or pravastatin for 72 h with IL2. NK cells incubated with the solvent of either fluvastatin or pravastatin at the same molar concentration as in statins incubated samples were the controls of statins treated cultures. At the end of the treatment cholesterol/cholesteryl ester was quantified using colorimetric methods according to the kit instruction. Results are shown as mg/106cells. Calcium Mobilization Assay To evaluate the kinetics of intracellular free calcium increase, NK cells were loaded for 1 h at 37uC with the calcium probe Fura-2AM, washed and stained with antibody to a given receptor at 4uC and then i analyzed after the addition of GAM to achieve optimal cross-linking. Samples were placed in a 1 cm quartz cuvette and Fura2 was IMR-1 excited at 340 nm and 380 nm; emitted light was filtered at 510 nm and fluorescence was monitored with the spectrofluorimeter LS50B. The ratio of 340/ 380 nm excitation Fura-2 emitted fluorescence at 510 nm was analyzed and nM i calculated by the computer program FLWinLab 3.0. To determine the percentage of responding cells to a given stimulus NK cells were labelled with Analysis of Activation of RhoA The level of activation of RhoA GTP-binding protein was analyzed on NK cells after LFA1 cross-linking. Briefly, 46106 NK cells for each time point were incubated with anti-LFA1 mAb for 30 min at 4uC and then cross-linked with GAM at 37uC in complete medium. Activation of RhoA was analysed using the RhoA activation kit at 5 min, 12 min and 30 min after cross-linking and HMG-CoA Reductase Inhibitors and NK Cell Cytolysis each sample was processed as indicated by manufacturer’s instruction. Each sample was performed in triplicates and the activation of RhoA in LFA1 cross-linked samples was evaluated in comparison to the level of activation in the absence of crosslinking. In parallel experiments, the actin distribution in a given cell was analyzed by confocal microscopy, after cross-linking of LFA1, with Alexa488 phalloidin. Analysis of Activation of akt1/PKB The activation of the serine/threonine kinase akt1/PKB in cell lysates of NK cells was assessed with a commercial ELISA assay kit. Akt1/PKB activity was tested upon ligation of surface receptors and the protein content in each sample was normalized. The amount of total akt1 and phosphorylated akt1was evaluated with specific antibodies at least in triplicate and results are expressed as % of akt1/PKB phosphorylat