Samples were analysed using negative electrospray ionization (ESI). The ion spray voltage was set at −4500 V. The source temperature was Adriamycin in vivo set at 400°C. Nitrogen was used as the nebulizer and auxiliary gas and was set at 20, 50 and 50 arbitrary units for the curtain gas, the ion source gas 1 and the ion source gas 2, respectively. MS/MS spectra of

15-epi-LXA4 showed the same fragmentation pattern as the published [31] and commercial source (data not shown) spectra. Moreover, LC-MS/MS analysis confirmed 15-epi-LXA4 stability and no changes in height peak and area were observed during the time of the in-vitro assay conditions and using the 15-epi-LXA4 concentration reported to show biological activity (data not shown). The synthetic www.selleckchem.com/products/mi-503.html peptide WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2) was purchased from Tocris Bioscience (Bristol, UK). IL-8 was purchased from Peprotech (Rocky Hill, NJ, USA). Montelukast, MK-571, compound 43 and SCH527123 were synthesized at the Medicinal Chemistry Department in Almirall R&D Centre (Sant Feliu de Llobregat, Barcelona, Spain). Human Chinese

hamster ovary (CHO)-FPR2/ALX (ES-610-C) and human CHO-CysLT1 (ES-470-C) cell lines were purchased from Perkin Elmer (Waltham, MA, USA). Surface expression of the receptor FPR2/ALX was monitored by flow cytometry using a commercial monoclonal antibody against the receptor. Results clearly show high levels of receptor expression in FPR2/ALX-recombinant CHO cells compared to non-transfected CHO cells (increased 40-fold in mean expression). In addition, information on Bmax of recombinant cell lines by a radioligand saturation binding assay was provided by Perkin Elmer Ribonuclease T1 and confirmed activity of both receptors in the recombinant cells. Ham’s F12 culture medium supplemented with 100 IU/ml penicillin and 400 μg/ml G418 was used to grow the cells. FPR2/ALX cell membrane preparation was performed from FPR2/ALX stable transfected CHO cells purchased from Perkin-Elmer. Adherent-growing CHO-h FPR2/ALX cells were washed in cold phosphate-buffered saline (PBS), harvested by scraping

and collected by centrifugation at 1500 g for 5 min. The cell pellet was washed twice with cold PBS and resuspended in homogenization buffer [15 mM Tris-HCl, pH 7·5, 2 mM MgCl2, 0·3 mM ethylenediamine teraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA)]. The cells were then lysed with an Ultraturrax homogenizer. Intact cells and nuclei were removed by centrifugation at 1000 g for 5 min. The cell membranes in the supernatant were then pelleted by centrifugation at 40 000 g for 25 min and resuspended in storage buffer (50 mM Tris-HCl pH 7·4, 0·5 mM EDTA, 10 mM MgCl2, 10% sucrose), aliquoted, quick-frozen in liquid N2 and stored at −80°C. Protein concentration in membrane preparations was determined using the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA).