Addendum: Resolving Inflammation: Synthesis, Configurational Assignment, and Biological Evaluations of RvD1 _{n −3 DPA}

The authors wish to provide detailed protocol that clarifies and supports their results with RvD1n−3 DPA and RvD1 in this manuscript. The following paragraph should be inserted at Line 3 on page 1479: A) Validation of n−3 DPA Resolvins The structural integrity of RvD1n−3 DPA and RvD1n−3 DPA ethyl ester were each verified using LC-MS-MS before bioassays and receptor assay. B) Activation of human ALX and GPR32: comparison of RvD1, RvD1n−3 DPA and RvD1n−3 DPA ethyl ester using GPCR-beta-arrestin based system: a step-by-step procedure (1) Ligand receptor interactions were monitored using the PathHunter® β-arrestin cell-based assays (Eurofins DiscoverX Corporation, Fremont, CA, USA) essentially as described earlier.[1] CHO-β-arrestin-GPR32 cells and HEK-β-arrestin-FPR2/ALX cells were purchased from DiscoverX, and maintained in HAM F-12 and DMEM media, respectively, supplemented with 10% FBS, 1X pen/strip, G418 (0.8 mg ml−1) and hygromycin (0.3 mg ml−1). (2) The day before each experiment, cells were plated onto 96-well plates (white with clear bottom; Costar #3903) at 20 000 cells per well. (3) The next day, the media was aspirated and cells were suspended in 90 microliters of PBS +/+ (with Ca2+ and Mg2+). Stocks of each compound (10−4 M) were used to prepare working solutions at 10−7 to 10−13 M in PBS +/+ by serial dilutions. (4) Ten microliters of each concentration was then added to each well to make final concentrations of 10−8 to 10−14 M. For vehicle controls, 10 microliters of PBS+/+ was added to the wells. Each concentration of each compound was tested in 3 to 4 replicates in 3 independent experiments. Assay plates were incubated at 37 °C and 5 % CO2 for 90 min. (5) DiscoverX PathHunter Detection Reagent (Eurofins DiscoverX Corporation, Fremont, CA, USA) was then used following the manufacturer's instruction; namely, 55 microliters of Working Detection Solution (mixture of Cell Assay Buffer: Substrate Reagent 1: Substrate Reagent 2; 19:5:1) was added to each well, and assay plates incubated in the dark at room temperature for 60 min. Chemiluminescence was then determined using a luminescence plate reader (SpectraMax M3; Molecular Device) at 0.5 s per well, collecting signals from all wavelengths as recommended by the manufacturer. Results are expressed as % increase of RLU (Relative luminescence) above vehicle controls. The 10 % increases represent 35.3±8.6 and 170.0±7.1 RLU arbitrary units for GPR32 and ALX, respectively. Note that the receptor denoted ALX/FPR2 was demonstrated bias in intracellular signaling with specific ligands and with pro-resolving mediator ligands,[2] confirmed in >175 publications in PubMed to date. References 1. S. Krishnamoorthy, A. Recchiuti, N. Chiang, S. Yacoubian, C. H. Lee, R. Yang, N. A. Petasis, C. N. Serhan, Proc. Natl. Acad. Sci. USA, 2010, 107, 1660. 2. J. G. Filep, Proc. Natl. Acad. Sci. USA, 2013, 110, 18033.