Aging Aging

318 Mariani, Alonso, and Solana[(test release — spontaneous release) /(maximum release – spontaneous release)] × 100.3.4. Analysis of Phosphoinositide Turnoverin NK Cells from Elderly People1. Labeling of inositol phospholipids: Resuspend purified NK lymphocytes at a concentrationof 1 × 10 6 cells in a final volume of 1 mL of inositol-free RPMI 1640supplemented with 10% heat-inactivated and dialyzed FCS. NK lymphocytes aremetabolically labeled with myo-[ 3 H] inositol (5 µCi/10 6 cells/mL) for 18 h at37°C in 5% CO 2 . Wash the labeled cells 2× for 10 min at 300g using cold inositol-freeRPMI 1640 with 10% FCS and resuspend the pellet in the same mediumat 2.5 × 10 6 cells/mL. Stimulate the purified NK cell samples (2.5 × 10 5 /100 µL)in Eppendorf tubes with either a similar number of target cells (E/T ratio 1:1) orwith an appropriate amount of MAb for various time intervals (from 1 to 30 min)at 37°C in a water bath. Incubate control samples for the same time both in theabsence of stimuli and with nonsusceptible targets and/or irrelevant MAbs. Stopincubation by adding methanol/chloroform/HCl 37% (80:160:1 v/v), keep thesamples at 4°C, and extract phospholipids by vortex-mixing the cells in the aboveextraction solution. After centrifugation (100,000g for 5 min) an upper aqueousphase containing inositol phosphates and a lower chloroform phase containingphospholipids are obtained. Collect the upper phase, dry it in vacuo, and store at–80°C until further use. Wash the lower phase with 0.7 mL of methanol/chloroform/1N HCl (235:15:245 v/v) by vigorous mixing. After centrifugation(100,000g for 5 min) collect the new lower phase and dry it in vacuo.2. Analysis of inositol phosphate (IP) fractions (IP, IP 2 , IP 3 ). To analyze inositolphosphates, treat the dried samples of the inorganic upper phase with 1 M KOH,resuspend these samples in 4 mL of bidistilled water, and load them onto Amprepion-exchange minicolumns. IP and IP 2 are eluted together from the column with5 mL of 0.1 M KHCO 3 , while the remaining IP 3 is eluted using a gradient based on0.17 M of KHCO 3 and 0.25 M KHCO 3 buffers at a flow rate of 1 mL/min. Collectsamples in three fractions of 5 mL and measure 3 H-labeled inositol phosphates byliquid scintillation counting using a β-counter. Compare the eluted peaks to retentiontimes for standards prepared from [ 3 H]PI, [ 3 H]PIP, and [ 3 H]PIP 2 . The scintillationcounting is set to obtain a counting error lower than 5%.3. Analysis of phosphatidylinositol phosphate fractions (PIP, PIP 2 , PIP 3 ). Solubilizethe lower phase containing phosphatidylinositols in chloroform/methanol(2:1) and spot it onto 1% K oxalate-sprayed TLC plates to separate [3H] labeledphosphatidylinositols. Develop TLC plates with chloroform/methanol/water/saturatedammonia (45:35:8:2 v/v), spray with Enhancer, and fluorograph at –80°C.Autoradiograph TLC plates before exposure to iodine to visualize internal lipidstandards obtained from Sigma (St. Louis, MO, USA). Scrape off the single“spot” of PI, PIP, and PIP 2 ; extract with 1.5 mL of 0.6 N HCl/methanol (60:40 v/v)for 48 h with gentle stirring, and count with a liquid scintillation counter using