Chemotaxis of PMN

Polymorph nuclear cells were prepared from citrated blood of healthy donors by a combination of sedimentation and Ficoll density gradient centrifugation as described (10). Sedimented cells were incubated with ice-cold aqua dest for lysis of erythrocytes, washed in ice-cold phosphate buffered saline (PBS), and suspended in PBS at 4 × 10⁶ cells/mL. To determine the effect of the PI3Kδ inhibitor LAS191954 on neutrophilic chemotaxis, stock solutions of the inhibitor stored at −80°C (prepared in DMSO at different concentrations starting at 3 × 10⁻³ M and following a fourfold bank dilution) were further diluted 1:500 in PBS containing 1% BSA to a twofold assay concentration and tested in a slightly modified chemotaxis assay (32). In brief, interleukine (IL)-8 (6 nM; Peprotech, Hamburg, Germany), fMLP (10 nM), and human recombinant C5a (12 nM) were diluted in PBS with Ca²⁺, Mg²⁺/0.1% BSA and added to the bottom wells of the chamber. After covering the bottom wells with a polycarbonate membrane (pore size: 5 µm, Costar Nucleopore GmbH, Tübingen, Germany), the top wells were filled with 1 × 10⁵ freshly prepared neutrophils in PBS with Ca²⁺, Mg²⁺/1% BSA that were preincubated with different concentrations of the PI3Kδ inhibitor LAS191954 (10 min, 37°C). After incubation for 1 h at 37°C, migration was terminated by removing the cells from the top wells. Migrated cells were transferred from the bottom wells to a microtiter plate and lysed with 0.1% hexabromide solution. Number of migrated neutrophils was determined via endogenous myeloperoxidase activity by adding myeloperoxidase substrate tetramethylbenzidine (Life Technologies, Paisley, UK). The redox reaction was stopped by 0.9 M sulfuric acid and oxidized tetramethylbenzidine measured at λ = 450 nm. The number of migrated neutrophils was calculated from a standard curve of cell lysates run in parallel.