Formoterol fumarate dehydrate

Formoterol fumarate dehydrate and salbutamol hydrochloride were purchased from Sigma-Aldrich (Pty) Ltd. (Johannesburg, South Africa), while indacaterol maleate was provided by Novartis International AG, Basel, Switzerland. All 3 agents were dissolved to a stock concentration of 10 mM in dimethylsulfoxide (DMSO) and used at final concentrations of 1, 10, and 100 nM in most of the assays described below and at 1–1000 nM in others. The local concentration of formoterol following inhalation of 24 μg of this agent has been estimated to be approximately 11 nM [16 (link)]. The final concentration of DMSO in each assay was 0.1% and appropriate solvent controls were included with each experimental system. Unless indicated, all other chemicals and reagents were purchased from Sigma-Aldrich.
Neutrophils were activated with the chemoattractant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 μM final) either alone or in combination with cytochalasin B (CB, 1 μM final) in assays of oxygen consumption and degranulation. An additional chemoattractant, platelet-activating factor (PAF, 200 nM final) was used in a more limited series of experiments.

Lipopolysaccharide (from E. coli serotype 0111:B4), formoterol fumarate dehydrate, DMSO, acetylcholine and histamine were purchased from Sigma (St. Louis, MO, USA). Roswell Park Memorial Institute 1640 medium, penicillin-streptomycin stabilized solution, and L-glutamine were purchased from Eurobio Biotechnology (Les Ulis, France). Roflumilast and roflumilast-N-oxide were synthesized by Nycomed GmbH (Konstanz, Germany). All other chemicals were of analytical grade and were obtained from Prolabo (Fontenay sous Bois, France). All cell culture plastics were from CCL (Nemours, France). Stock solutions of the PDE4 inhibitors were prepared in DMSO at 10 mM. A 2.5 mM formoterol stock solution was prepared in 10% DMSO. All subsequent dilutions were prepared daily in complete culture medium.