136 Exp. Mol. Med. Vol. 44(2), 130-137, 2012 Probes (Eugene, OR). PTX was purchased from Calbio- chem (San Diego, CA). Isolation of leukocytes The Institutional Review Board (IRB) of Ajou University Hospital specifically approved this study (approval ID: AJIRB-MED-SMP-10-074). We obtained written informed consent from all participants involved in this study. Peripheral blood leukocytes were isolated from healthy donors. Human neutrophils were isolated according to the standard procedures of dextran sedimentation, hypotonic lysis of erythrocytes, and a lymphocyte separation medium gradient as described previously (Bae et al., 2001). PBMCs were separated on a Histopaque-1077 gradient. After two washings with HBSS without Ca2+ and Mg2+, the PBMCs were suspended in 10% FBS containing RPMI 1640 and incubated for 60 min at 37oC to allow attachment of monocytes to the culture dish. The cells were washed five times with warm RPMI medium to washout lympho- cytes, after which the attached monocytes were collected as described previously (Bae et al., 2001). The isolated hu- man leukocytes were then used promptly. Cell culture NIH3T3 (NIH Swiss mouse embryo fibroblasts), 3Y1 (Rat embryonic fibroblasts), 3T3L1 (preadipocytes), and PC12 (rat adrenal pheochromocytoma) cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained as recommended. The cells were maintained at about 1 × 106 cells/ml under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37oC). Initial screening of PS-SPCLs and measurement of intracellular Ca2+ increase The hexapeptide libraries were obtained from the Peptide Library Support Facility of Pohang University of Science and Technology as described previously (Bae et al., 2001). Finally, 114 peptide pools (Cys was excluded in the con- struction of the libraries) were individually dissolved in wa- ter at a final concentration of 27 nM per peptide sequence in each pool. For initial screening of the PS-SPCLs, we measured the level of [Ca2+]i by using Grynkiewicz’s meth- od with fura-2/AM (Grynkiewicz et al., 1985). Briefly, pre- pared cells were incubated with 3 μM fura-2/AM at 37oC for 50 min in fresh serum-free RPMI 1640 medium under con- tinuous stirring. Then, 2 × 106 cells were aliquoted for each assay in Ca2+-free Locke's solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 5 mM HEPES, pH 7.3, 10 mM glucose, and 0.2 mM EGTA) and incubated with the in- dicated concentrations of peptide pools (final 50 pM per peptide sequence for the initial screening). Fluorescence changes at the dual excitation wavelengths of 340 nm and 380 nm and the emission wavelength of 500 nm were measured, and the calibrated fluorescence ratio was trans- lated into [Ca2+]i. Chemotaxis assay Chemotaxis assays were performed using multiwell cham - bers (Neuroprobe Inc., Gaithersburg, MD) (Bae et al., 2001). Briefly, prepared human neutrophils were sus- pended in RPMI 1640 at a concentration of 1 ×106 cells/ml, and 25 μl of the suspension was placed into the upper well of a chamber separated by a 3 μm polyhydrocarbon filter from the lower well containing peptides. After incubation for 90 minutes at 37oC, non-migrated cells were removed by scarping, and cells that migrated across the filter were de- hydrated, fixed, and stained with hematoxylin (Sigma, St. Louis, MO). Stained cells in five randomly chosen high power fields (400 X) in the well were then counted (Bae et al., 2001). Measurement of superoxide anion production Superoxide anion generation was determined by measur- ing cytochrome c reduction using a microtiter 96-well plate ELISA reader (Bio-Tek instruments, EL312e, Winooski, VT), as previously described (Bae et al., 2001). Human neutrophils (2 × 106 cells in RPMI 1640 medium) were pre- incubated with 50 μM cytochrome c at 37oC for 5 min and subsequently incubated with each peptide. Superoxide generation was determined by measuring light absorption changes at 550 nm over 5 min at 1 min intervals. Statistical analysis The results are expressed as mean ± S. E. of data ob- tained from the indicated number of experiments per- formed. Statistical significance was determined using the Student t test. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0020940), by National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2009 0093198), and Samsung Research Fund, Sungkyunkwan University, 2011. References Aramburu J, Yaffe MB, Lopez-Rodriguez C, Cantley LC, Hogan PG, Rao A. 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